Vector

ABSTRACT

A vector comprising a nucleotide sequence of interest (“NOI”) encoding a product of interest (“POI”) is described. The NOI and/or the POI is capable of recognizing a tumor, such that in use the vector is capable of delivering the NOI and/or the POI to the tumor.

[0001] The present invention relates to a vector, preferably for use inmedicine.

[0002] As it is well known in the art, a vector is a tool that allows orfaciliates the transfer of an entity from one environment to another. Byway of example, some vectors used in recombinant DNA techniques allowentities—such as a segment of DNA (such as a heterologous DNA segment,such as a heterologous cDNA segment)—to be transferred into a targetcell. Optionally, once within the target cell, the vector may then serveto maintain the heterologous DNA within the cell or may act as a unit ofDNA replication. Examples of vectors used in recombinant DNA techniquesinclude plasmids, chromosomes, artificial chromosomes or viruses.

[0003] Thus, vectors can be used to deliver proteins and/or nucleotidesequences to targeted cells, such as tumour cells.

[0004] However, as it is well known, nucleotide sequences and proteinsare complex molecules which may be produced from biological sources,most usually from genetically engineered organisms or cell cultures.Furthermore, the procedures for the production of nucleotide sequencesand proteins can be complicated, labour intensive and costly.Furthermore, pharmacological properties and other aspects of thefunction of some proteins—such as immunoglobulins derived from non-humanbiological sources—and nucleotide sequences may frequently differ inimportant ways from the activity of the corresponding natural humanimmunoglobulins produced in human cells. By way of backgroundinformation, an immunoglobulin is a member of a family of relatedmultimeric proteins which are normally secreted from cells of theB-lymphocyte lineage of a vertebrate, whose typical function is to bindspecifically with a region of a macromolecule identified as non-self.Immunoglobulins represent a major component of the immune responserepertoire of the organism and are synonymous with “antibodies”.

[0005] One major cause of such differences in activity may be due tovariations in the pattern of glycosylation of proteins derived fromdifferent species (reviewed in Bebbington 1995; In MonoclonalAntibodies: the second generation ed. H. Zola pg 165-181). Furthermore,systemic administration of proteins (especially those containing toxindomains) and nucleotide sequences can induce additional pharmacokineticand toxicological problems (reviewed in Scheinberg and Chapman 1995. InMonoclonal antibodies (ed. Birch and Lennox) Chapter 2.1).

[0006] Thus, the present invention seeks to provide an improved vectorsystem for delivering a nucleotide sequence of interest and/or a productexpressed by the same.

[0007] According to a first aspect of the present invention there isprovided a vector comprising a nucleotide sequence (“NS”) coding for atumour interacting protein (“TIP”) and optionally comprising anucleotide sequence of interest (“NOI”) which NOI encodes a product ofinterest (“POI”): wherein the TIP is capable of recognising a tumour,such that in use the vector is capable of delivering the NOI and/or thePOI to the tumour.

[0008] According to a second aspect of the present invention there isprovided a method of delivering a nucleotide sequence of interest(“NOI”) and/or a product of interest (“POI”) encoded by same to atumour, wherein the NOI and/or POI are delivered to the tumour by use ofa vector comprising the NOI and/or expressing the POI; wherein the NOIand/or the POI is capable of recognising a tumour; wherein the NOIand/or the POI is delivered to the tumour; and wherein the vector is avector according to the present invention.

[0009] According to a third aspect of the present invention there isprovided the use of a vector to deliver a nucleotide sequence ofinterest (“NOI”) and/or a product of interest (“POI”) encoded by same toa tumour, wherein the NOI and/or POI are delivered to the tumour by useof the vector which comprises the NOI and/or expresses the POI; whereinthe NOI and/or the POI is capable of recognising a tumour when the NOIand/or the POI is delivered to the tumour; and wherein the vector is avector according to the present invention.

[0010] According to a fourth aspect of the present invention there isprovided a method of treating a subject in need of same, the methodcomprising delivering a nucleotide sequence of interest (“NOI”) and/or aproduct of interest (“POI”) encoded by same to a tumour, wherein the NOIand/or POI are delivered to the tumour by use of a vector comprising theNOI and/or expressing the POI; wherein the NOI and/or the POI is capableof recognising a tumour; wherein the NOI and/or the POI is delivered tothe tumour; and wherein the vector is a vector according to the presentinvention.

[0011] According to a fifth aspect of the present invention there isprovided the use of a genetic vectors to deliver a therapeutic geneencoding a TIP—preferably a tumour binding protein (“TBP”)—morepreferably a secretable TIP (preferably a secretable TBP)—to theinterior of a tumour mass.

[0012] According to a sixth aspect of the present invention there isprovided a gene delivery system for targeting one or more genes encodinga TIP (preferably a TBP) to a tumour, comprising a genetic vectorencoding a TIP (preferably a TBP) and an in vivo gene-delivery system.

[0013] According to a seventh aspect of the present invention there isprovided a method of treating cancer comprising administering a TIP(preferably a TBP) gene or genes in a gene delivery system according tothe present invention either systemically or directly to the site of atumour.

[0014] According to an eighth aspect of the present invention there isprovided a gene delivery system for introducing one or more genesencoding a TIP preferably a TBP) into cells of the haematopoietic(preferably myeloid haematopoietic) cell lineage either in vivo or exvivo.

[0015] According to a ninth aspect of the present invention there isprovided a method for treating cancer in a mammal, comprisingadministering to an individual a gene delivery system according to thepresent invention that is capable of expressing a TBP in cells derivedfrom a haematopoietic (preferably myeloid haematopoietic) origin.

[0016] According to a tenth aspect of the present invention there isprovided a genetic vector comprising a therapeutic gene or genesencoding a TIP (preferably a TBP), operably linked to an expressionregulatory element selectively functional in a cell type present withina tumour mass.

[0017] According to an eleventh aspect of the present invention there isprovided a genetic vector comprising a therapeutic gene or genes isdelivered to the interior of the tumour wherein the therapeutic geneencodes a TIP (preferably a TBP), which additionally contains one ormore effector domains.

[0018] According to a twelfth aspect of the present invention there isprovided a method of treating cancer in a mammal which comprisesadministering to an individual a combination of a cytokine or acytokine-encoding gene and one or more TIP (preferably a TBP) genesaccording to any of the previous aspects of the invention.

[0019] According to a thirteenth aspect of the present invention thereis provided the delivery of TIP—(preferably a TBP—) encoding genes tothe site of a tumour.

[0020] Preferably the vector comprises the NOI.

[0021] In one preferred aspect, the vector is expressing the POI.

[0022] The vector of the present invention may be useful for inter aliamedical applications—such as diagnostic or therapeutic applications.

[0023] Preferably the NOI is a therapeutic NOI and/or the POI is atherapeutic POI.

[0024] On occasions in the following text, the NS and NOI may beindividually or collectively referred to as being a gene.

[0025] The NS and NOI can be any suitable nucleotide sequence. Forexample, independently they can be DNA or RNA—which may syntheticallyprepared or may be prepared by use of recombinant DNA techniques or maybe isolated from natural sources or may be combinations thereof. The NOImay be a sense sequence or an antisense sequence.

[0026] There may be a plurality of NSs or NOIs, which may be directly orindirectly joined to each other, or combinations thereof. Thus, theexpressed product may have two or more effector domains (which may bethe same or different) and/or two or more TIP domains (which may be thesame or different).

[0027] Preferably in use the vector is capable of delivering the NOIand/or the POI to the interior of a tumour mass.

[0028] In addition to cancerous cell, the cell types present within atumour mass include but are not limited to macrophages, lymphocytes,tumour infiltrating lymphocyes, endothelial cells etc.

[0029] Preferably the NS and/or the TIP comprises at least one tumourbinding domain capable of interacting with at least one tumourassociated cell surface molecule (“TACSM”).

[0030] In accordance with the present invention the TACSM can includebut is not limited to a cell surface molecule which plays a role intumour cell growth, migration or metastasis, a receptor for adhesiveproteins such as the integrin vitronectin receptor, a growth factorreceptor (such as epidermal growth factor (EGF) receptor,platelet-derived growth factor (PDGF) receptor, fibroblast-derivedgrowth factor (FDGF) receptor, nerve growth factor receptor,insulin-like growth factor (IGF-1) receptor; a plasminogen activator; ametalloproteinase (such as colllagenase) 5T4 antigen; a tumour specificcarbohydrate moiety; an oncofetal antigen; a mucin; a growth factorreceptor; a glycoprotein; and an antigen restricted in its tissuedistribution.

[0031] Preferably the TACSM is selectively expressed on one cell type oron a restrictive number of cell types.

[0032] Preferably in use the vector is capable of delivering the NOIand/or the POI to a selective tumour site.

[0033] Preferably the TIP is or comprises a tomour binding protein(“TBP”).

[0034] Preferably the TIP is a TBP.

[0035] Examples of a TBP include: an adhesion molecule such asIntercellular adhesion molecule, ICAM-1, ICAM-2, LFA-1, LFA-2, LFA-3,LECAM-1, VLA-4, ELAM, N-CAM, N-cadherin, P-Selectin, CD44 and itsvariant isoforms (in particular CD44v6, CD44v7-8), CD56; a growth factorreceptor ligand such epidermal growth factor (EGF), Platelet-derivedgrowth factor (PDGF), Fibroblast-derived growth factor (FDGF), Nervegrowth factor, vasopressin, insulin, insulin-like growth factor (IGF-1),hepatocyte growth factor, nerve growth factor, human growth factor,brain derived growth factor, ciliary neutrophic factor, glial cellline-derived growth factor; heavy and light chain sequences from animmunoglobulin (Ig) variable region (from human and animal sources),engineered antibody or one from a phage display library. A phage displaylibrary is a technique of expressing immunoglobulin genes inbacteriophage has been developed as a means for obtaining antibodieswith the desired binding specificities. Expression systems, based onbacteriophage lambda, and more recently filamentous phage have beendeveloped. The bacteriophage expression systems can be designed to allowheavy and light chains to form random combinations which are tested fortheir ability to bind the desired antigen.

[0036] The TBP may contain an effector domain which is activated onbinding of the TPB to the TASCM. The effector domain or momains may beactivated on binding of the TBP to a TASCM leading to inhibition oftumour cell proliferation, survival or dissemination. The effectordomain may possess enzymatic activity (such as a pro drug activatingenzyme) or the effector domain may include a toxin, or an immuneenhancer, such as a cytokine/lymphokine such as those listed above.

[0037] Preferably the TBP comprises one or more binding domains capableof interacting with one or more TACSMs which are present on thecancerous cells—which TACSMs may be the same or different.

[0038] The term “interacting” includes direct binding, leading to abiological effect as a result of such binding.

[0039] Preferably the TIP is or comprises at least part of an antibody.

[0040] As is well known, antibodies play a key role in the immunesystem. In brief, the immune system works in three fundamentallydifferent ways: by humoral immunity, by cellular immunity and bysecretion of stimulatory proteins, called lymphokines. Humoral immunityrelies on proteins collectively called immunoglobulin which constituteabout 20% of the proteins in the blood. A singly immunoglobulin moleculeis called an antibody but “antibody” is also used to mean many differentmolecules all directed against the same target molecule. Humoralimmunity also involves complement, a set of proteins that are activatedto kill bacteria both nonspecifically and in conjunction with antibody.

[0041] In cellular immunity, intact cells are responsible forrecognition and elimination reactions. The body's first line of defenseis the recognition and killing of microorganisms by phagocytes, cellsspecialised for the ingestion and digestion of unwanted material. Thesecells include neutrophils and macrophages. A key role of antibodies isto help phagocytes recognise and destroy foreign materials.

[0042] In order to perform these functions, the antibody is divided intotwo regions: binding (Fab) domains that interact with the antigen andeffector (Fc) domains that signal the initiation of prcesses such asphagocytosis. Each antibody molecule consists of two classes ofpolypeptide chains, light (L) chains and heavy (H) chains. A singleantibody has two indentical copies of the L chain and two of the Hchain. The N-terminal domain from each chain forms the variable regions,which constitute the antigen-binding sites. The C-terminal domain iscalled the constant region. The variable domains of the H (V_(H)) and L(V_(L)) chains consitute an Fv unit and can interact closely to form asingle chain Fv (ScFv) unit. In most H chains, a hinge region is found.This hinge region is flexible and allows the Fab binding regions to movefreely relative to the rest of the molecule. The hinge region is alsothe place on the molecule most susceptible to the action of proteasewhich can split the antibody into the antigen binding site (Fab) and theeffector (Fc) region.

[0043] The domain structure of the antibody molecule is favourable toprotein engineering, facilitating the exchange between molecules offunctional domains carrying antigen-binding activities (Fabs and Fvs) oreffector functions (Fc). The structure of the antibody also makes iteasy to produce antibodies with an antigen recognition capacity joinedto molecules such as toxins, lymphocytes or growth factors.

[0044] Monoclonal antibodies are homogenous antibodies of the sameantigenic specificity representing the product of a single clone ofantibody-producing cells. It was recognised that monoclonal antibodiesoffered the basis for human therapeutic products. However, althoughmouse antibodies are similiar to human antibodies, they are sufficientlydifferent that they are recognised by the immune system as foreignbodies, thereby giving rise to an immunological response. Thishuman-anti-mouse-antibody (HAMA) response limits the usefulness of mouseantibodies as human therapeutic products.

[0045] Chimeric antibody technology involves the transplantation ofwhole mouse antibody variable domains onto human antibody constantdomains. Chimeric antibodies are less immunogenic than mouse antibodiesbut they retain their antibody specificity and show reduced HAMAresponses.

[0046] In chimeric antibodies, the variable region remains completelymurine. However, the structure of the antibody makes it possible toproduce variable regions of comparable specificity which arepredominantly human in origin. The antigen-combining site of an antibodyis formed from the six complementarity-determining regions (CDRs) of thevariable portion of the heavy and light chains. Each antibody domainconsists of seven antiparallel β-sheets forming a β-barrel with loopsconnecting the β-strands. Among the loops are the CDR regions. It isfeasible to more the CDRs and their associated specificity from onescaffolding β-barrel to another. This is called CDR-grafting.CDR-grafted antibodies appear in early clinincal studies not to be asstrongly immunogenic as either mouse or chimaeric antibodies. Moreover,mutations may be made outside the CDR in order to increase the bindingactivity thereof, as in so-called humanised antibodies.

[0047] Fab, Fv, and single chain Fv (ScFv) fragments with VH and VLjoined by a polypeptide linker exhibit specificities and affinities forantigen similiar to the original monoclonal antibodies. The ScFv fusionproteins can be produced with a nonantibody molecule attached to eitherthe amino or carboxy terminus. In these molecules, the Fv can be usedfor specific targeting of the attached molecule to a cell expressing theappropriate antigen. Bifunctional antibodies can also be created byengineering two different binding specificities into a single antibodychain. Bifunctional Fab, Fv and ScFv antibodies may comprise engineereddomains such as CDR grafted or humanised domains.

[0048] In virally directed enzyme therapy (VDEPT), a foreign gene isdelivered to normal and cancerous cells by a viral vector—such as aretroviral vector. The foreign gene codes for an enzyme that can converta non-toxic prodrug (eg 5-fluorocytosine) to a toxic metabolite(5-fluorouracil) that will kill those cells making it (Sikora et al 1994Ann New York Acad Sci 71b: 115-124). If the promoter utilised is tumourspecific, then the toxic product will only be synthesised in the tumourcells. Studies in animal models have demonstrated that this type oftreatment can deliver up to 50-fold more drug than by conventional means(Connors and Knox 1995 1995 Stem Cells 13: 501-511). A variation of thistechnique uses tumour associated antibodies conjugated to prodrugconverting enzymes to provide specific delivery to tumours. This methodis referred to as antibody-directed enzyme prodrug therapy (ADEPT)(Maulik S and Patel S D “Molecular Biotechnology” 1997 Wiley-Liss Inc pp45).

[0049] A large number of monoclonal antibodies and immunoglobulin-likemolecules are known which bind specifically to antigens present on thesurfaces of particular cell types such as tumour cells. Procedures foridentifying, characterising, cloning and engineering these molecules arewell established, for example using hybridomas derived from mice ortransgenic mice, phage-display libraries or scFv libraries. Genesencoding immunoglobulins or immunoglobulin-like molecules can beexpressed in a variety of heterologous expression systems. Largeglycosylated proteins including immunoglobulins are efficiently secretedand assembled from eukaryotic cells, particularly mammalian cells.Small, non-glycosylated fragments such as Fab, Fv, or ScFv fragments canbe produced in functional form in mammalian cells or bacterial cells.

[0050] The immunoglobulin or immunoglobulin-like molecule may be derivedfrom a human antibody or an engineered, humanised rodent antibody suchas a CDR-grafted antibody or may be derived from a phage-display libraryor may be a synthetic immunoglobulin-like molecule.

[0051] The antigen-binding domain may be comprised of the heavy andlight chains of an immunoglobulin, expressed from separate genes, or mayuse the light chain of an immunoglobulin and a truncated heavy chain toform a Fab or a F(ab)′₂ fragment. Alternatively, truncated forms of bothheavy and light chains may be used which assemble to form a Fv fragment.An engineered scFv fragment may also be used, in which case, only asingle gene is required to encode the antigen-binding domain. In oneprefered aspect, the antigen-binding domain is formed from a Fv or ascFv.

[0052] When a pathogen invades the body, lymphocytes respond with threetypes of reaction The lymphocytes of the humoral system (B cells)secrete antibodies that can bind to the pathogen, signalling itsdegradation by macrophages and other cells. The lymphocytes of thecellular system (T cells) carry out two major types of functions.Cytotoxic T lymphocytes (CTLs) develop the ability to directly recogniseand kill the cells infected by the pathogen. Helper T cells (TH cells)independently recognise the pathogen and secrete protein factors(lympholines) that stimulate growth and responsiveness of B cells, Tcells, and macrophages, thus greatly strengthening the power of theimmune response.

[0053] Thus, in one preferred aspect, the TIP comprises animmunoglobulin, or a part thereof, or a bioisostere thereof.

[0054] In a preferred embodiment, the TIP comprises IgG and/or IgE, or apart thereof, or a bioisostere thereof.

[0055] In a more preferred embodiment, the TIP comprises IgE, or a partthereof, or a bioisostere thereof.

[0056] Preferably the TIP recognises a trophoblast cell surface antigen.

[0057] Preferably the TIP recognises the 5T4 antigen.

[0058] The trophoblast cell surface antigen, originally defined bymonoclonal antibody 5T4 (Hole and Stern 1988 Br. J. Cancer 57; 239-246),is expressed at high levels on the cells of a wide variety of humancarcinomas (Myers et al. 1994 J. Biol. Chem. 269; 9319-9324) but, innormal tissues of non-pregnant individuals, is essentially restricted tolow level expression on a few specialised epithelia (Myers et al. ibid.and references therein). The 5T4 antigen has been implicated incontributing to the development of metastatic potential and thereforeantibodies specifically recognising this molecule may have clinicalrelevance in the treatment of tumours expressing the antigen.

[0059] The variable region of the 5T4 monoclonal antibody can also behumanised by a number of techniques, which are known in the art,including grafting of the CDR region sequences on to a human backbone.These can then be used to construct an intact humanised antibody or ahumanised single chain antibody (Sab), such as an ScFv coupled to an Fcregion (see Antibody Engineering: a practical approach, ed McCafferty etal. 1996 OUP).

[0060] Here the term Sab is not limited to just a human or a humanisedsingle chain antibody. Preferably, however the Sab is a human singlechaim anitibody or a humanised single chain antibody, or partthereof—such as ScFv coupled to an Fc region.

[0061] Preferably the NS and NOI and/or the TIP and POI are linkedtogether.

[0062] Preferably the TIP and POI are directly linked together.

[0063] Preferably any one or more of the NS, NOI, TIP, and POI furthercomprise at least one additional functional component.

[0064] Preferably, at least the TIP and/or POI further comprise at leastone additional functional component.

[0065] Preferably the additional functional component is selected fromany one or more of a signalling entity (such as a signal peptide), animmune enhancer, a toxin, or a biologically active enzyme.

[0066] In a preferred aspect the POI is a secretable POI. Thus, in thisaspect of the present invention, preferably, the additional functionalcomponent is at least an entity capable of causing the POI to besecreted—such as a signalling entity.

[0067] Another preferred additional component is a promoter.

[0068] The term “promoter” is used in the normal sense of the art, e.g.an RNA polymerase binding site in the Jacob-Monod theory of geneexpression.

[0069] Preferably the vector comprises a tumour specific promoterenhancer.

[0070] Other preferred additional components include entities enablingefficient expression of the POI. For example, the additional componentmay be an enhancer. Here, the term enhancer includes a DNA sequencewhich binds to other protein components of the transcription initiationcomplex and thus facilitates the initiation of transcription directed byits associated promoter.

[0071] Preferably the vector is used to deliver the NOI and/or POI exvivo and/or in vivo to the tumour.

[0072] The vector of the present invention is useful in gene therapy fordelivering the NOI and/or the POI to a selective site.

[0073] Gene therapy includes any one or more of: the addition, thereplacement, the deletion, the supplementation, the manipulation etc. ofone or more nucleotide sequences in, for example, one or more targetedsites—such as targeted cells. If the targeted sites are targeted cells,then the cells may be part of a tissue or an organ. General teachings ongene therapy may be found in Molecular Biology (Ed Robert Meyers, PubVCH, such as pages 556-558).

[0074] By way of further example, gene therapy also provides a means bywhich any one or more of: a nucleotide sequence, such as a gene, can beapplied to replace or supplement a defective gene; a pathogenic gene orgene product can be eliminated; a new gene can be added in order, forexample, to create a more favourable phenotype; cells can be manipulatedat the molecular level to treat cancer (Schmidt-Wolf and Schmidt-Wolf,1994, Annals of Hematology 69;273-279) or other conditions—such asimmune, cardiovascular, neurological, inflammatory or infectiousdisorders; antigens can be manipulated and/or introduced to elicit animmune response—such as genetic vaccination.

[0075] The vector of the present invention may be a viral vector or anon-viral vector. Non-viral delivery systems include but are not limitedto DNA transfection methods. Here transfection includes a process usinga non-viral vector to deliver a gene to a target mammalian cell. Typicaltransfection methods include electroporation, DNA biolistics,lipid-mediated transfection, compacted DNA-mediated transfection,liposomes, immunoliposomes, lipofectin, cationic agent-mediated,cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556),and combinations thereof. Viral delivery systems include but are notlimited to adenovirus vector, an adeno-associated viral (AAV) vector, aherpes viral vector, retroviral vector, lentiviral vector, baculoviralvector. Other examples of vectors include ex vivo delivery systems—whichinclude but are not limited to DNA transfection methods such aselectroporation, DNA biolistics, lipid-mediated transfection, compactedDNA-mediated transfection).

[0076] Preferably the vector is a viral vector.

[0077] Preferably the vector is a retroviral vector.

[0078] In recent years, retroviruses have been proposed for use in genetherapy. Essentially, retroviruses are RNA viruses with a life cycledifferent to that of lytic viruses. In this regard, when a retrovirusinfects a cell, its genome is converted to a DNA form. In slightly moredetail, a retrovirus is a virus which contains genomic RNA which onentry into a host cell is converted to a DNA molecule by a reversetranscriptase enzyme. The DNA copy serves as a template for theproduction of new RNA genomes and virally encoded proteins necessary forthe assembly of infectious viral particles. Thus, a retrovirus is aninfectious entity that replicates through a DNA intermediate.

[0079] There are many retroviruses and examples include: murine leukemiavirus (MLV), human immunodeficiency virus (HIV), equine infectiousanaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcomavirus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemiavirus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murinesarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avianmyelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus(AEV).

[0080] A detailed list of retroviruses may be found in Coffin et al(“Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: J MCoffin, S M Hughes, H E Varmus pp 758-763).

[0081] Details on the genomic structure of some retroviruses may befound in the art. By way of example, details on HIV may be found fromthe NCBI Genbank (i.e. Genome Accession No. AF033819).

[0082] All retroviruses contain three major coding domains, gag, pot,env, which code for essential virion proteins. Nevertheless,retroviruses may be broadly divided into two categories: namely,“simple” and “complex”. These categories are distinguishable by theorganisation of their genomes. Simple retroviruses usually carry onlythis elementary information. In contrast, complex retroviruses also codefor additional regulatory proteins derived from multiple splicedmessages.

[0083] Retroviruses may even be further divided into seven groups. Fiveof these groups represent retroviruses with oncogenic potential. Theremaining two groups are the lentiviruses and the spumaviruses. A reviewof these retroviruses is presented in “Retroviruses” (1997 Cold SpringHarbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmus pp1-25).

[0084] All oncogenic members except the human T-cell leukemiavirus-bovine leukemia virus group (HTLV-BLV) are simple retroviruses.HTLV, BLV and the lentiviruses and spumaviruses are complex. Some of thebest studied oncogenic retroviruses are Rous sarcoma virus (RSV), mousemammary tumour virus (MMTV) and murine leukemia virus (MLV) and thehuman T-cell leukemia virus (HTLV).

[0085] The lentivirus group can be split even further into “primate” and“non-primate”. Examples of primate lentiviruses include the humanimmunodeficiency virus (HIV), the causative agent of humanauto-immunodeficiency syndrome (AIDS), and the simian immunodeficiencyvirus (SIV). The non-primate lentiviral group includes the prototype“slow virus” visna/maedi virus (VMV), as well as the related caprinearthritis-encephalitis virus (CAEV), equine infectious anaemia virus(EIAV) and the more recently described feline immunodeficiencey virus(FIV) and bovine immunodeficiencey virus (BIV).

[0086] A distinction between the lentivirus family and other types ofretroviruses is that lentiviruses have the capability to infect bothdividing and non-dividing cells (Lewis et al 1992 EMBO. J 11; 3053-3058,Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, otherretroviruses—such as MLV—are unable to infect non-dividing cells such asthose that make up, for example, muscle, brain, lung and liver tissue.

[0087] During the process of infection, a retrovirus initially attachesto a specific cell surface receptor. On entry into the susceptible hostcell, the retroviral RNA genome is then copied to DNA by the virallyencoded reverse transcriptase which is carried inside the parent virus.This DNA is transported to the host cell nucleus where it subsequentlyintegrates into the host genome. At this stage, it is typically referredto as the provirus. The provirus is stable in the host chromosome duringcell division and is transcribed like other cellular proteins. Theprovirus encodes the proteins and packaging machinery required to makemore virus, which can leave the cell by a process sometimes called“budding”.

[0088] As already indicated, each retroviral genome comprises genescalled gag, pol and env which code for virion proteins and enzymes.These genes are flanked at both ends by regions called long terminalrepeats (LTRs). The LTRs are responsible for proviral integration, andtranscription. They also serve as enhancer-promoter sequences. In otherwords, the LTRs can control the expression of the viral gene.Encapsidation of the retroviral RNAs occurs by virtue of a psi sequencelocated at the 5′ end of the viral genome.

[0089] The LTRs themselves are indentical sequences that can be dividedinto three elements, which are called U3, R and U5. U3 is derived fromthe sequence unique to the 3′ end of the RNA. R is derived from asequence repeated at both ends of the RNA and U5 is derived from thesequence unique to the 5′ end of the RNA. The sizes of the threeelements can vary considerably among different retroviruses.

[0090] For ease of understanding, a simple, generic diagram (not toscale) of a retroviral genome showing the elementary features of theLTRs, gag, pol and env is presented below.

[0091] For the viral genome, the site of transcription initiation is atthe boundary between U3 and R in the left hand side LTR (as shown above)and the site of poly (A) addition (termination) is at the boundarybetween R and U5 in the right hand side LTR (as shown above). U3contains most of the transcriptional control elements of the provirus,which include the promoter and multiple enhancer sequences responsive tocellular and in some cases, viral transcriptional activator proteins.Some retroviruses have any one or more of the following genes that codefor proteins that are involved in the regulation of gene expression:tat, rev, tax and rex.

[0092] As shown in the diagram above, the basic molecular organisationof a retroviral RNA genome is (5′) R-U5-gag, pol, env-U3-R (3′). In aretroviral vector genome gag, pol and env are absent or not functional.The R regions at both ends of the RNA are repeated sequences. U5 and U3represent sequences unique, respectively, to the 5′ and 3′ ends of theRNA genome. These three sets of end sequences go to form the longterminal repeats (LTRs) in the proviral DNA, which is the form of thegenome which integrates into the genome of the infected cell. The LTRsin a wild type retrovirus consist of (5′)U3-R-U5 (3′), and thus U3 andU5 both contain sequences which are important for proviral integration.Other essential sequences required in the genome for proper functioninginclude a primer binding site for first strand reverse transcription, aprimer binding site for second strand reverse transcription and apackaging signal.

[0093] With regard to the structural genes gag, pol and env themselvesand in slightly more detail, gag encodes the internal structural proteinof the virus. Gag is proteolytically processed into the mature proteinsMA (matrix), CA (capsid), NC (nucleocapsid). The gene pol encodes thereverse transcriptase (RT), which contains both DNA polymerase, andassociated RNase H activities and integrase (IN), which mediatesreplication of the genome. The gene env encodes the surface (SU)glycoprotein and the transmembrane (TM) protein of the virion, whichform a complex that interacts specifically with cellular receptorproteins. This interaction leads ultimately to fusion of the viralmembrane with the cell membrane.

[0094] The envelope protein is a viral protein which coats the viralparticle and plays an essential role in permitting viral entry into atarget cell. The envelope glycoprotein complex of retroviruses includestwo polypeptides: an external, glycosylated hydrophilic polypeptide (SU)and a membrane-spanning protein (TM). Together, these form an oligomeric“knob” or “knobbed spike” on the surface of a virion. Both polypeptidesare encoded by the env gene and are synthesised in the form of apolyprotein precursor that is proteolytically cleaved during itstransport to the cell surface. Although uncleaved Env proteins are ableto bind to the receptor, the cleavage event itself is necessary toactivate the fusion potential of the protein, which is necessary forentry of the virus into the host cell. Typically, both SU and TMproteins are glycosylated at multiple sites. However, in some viruses,exemplified by MLV, TM is not glycosylated.

[0095] Although the SU and TM proteins are not always required for theassembly of enveloped virion particles as such, they do play anessential role in the entry process. In this regard, the SU domain bindsto a receptor molecule—often a specific receptor molecule—on the targetcell. It is believed that this binding event activates the membranefusion-inducing potential of the TM protein after which the viral andcell membranes fuse. In some viruses, notably MLV, a cleavageevent—resulting in the removal of a short portion of the cytoplasmictail of TM—is thought to play a key role in uncovering the full fusionactivity of the protein (Brody et al 1994 J. Virol. 68: 4620-4627, Reinet al 1994 J. Virol. 68: 1773-1781). This cytoplasmic “tail”, distal tothe membrane-spanning segment of TM remains on the internal side of theviral membrane and it varies considerably in length in differentretroviruses.

[0096] Thus, the specificity of the SU/receptor interaction can definethe host range and tissue tropism of a retrovirus. In some cases, thisspecificity may restrict the transduction potential of a recombinantretroviral vector. Here, transduction includes a process of using aviral vector to deliver a non-viral gene to a target cell. For thisreason, many gene therapy experiments have used MLV. A particular MLVthat has an envelope protein called 4070A is known as an amphotropicvirus, and this can also infect human cells because its envelope protein“docks” with a phosphate transport protein that is conserved between manand mouse. This transporter is ubiquitous and so these viruses arecapable of infecting many cell types. In some cases however, it may bebeneficial, especially from a safety point of view, to specificallytarget restricted cells. To this end, several groups have engineered amouse ecotropic retrovirus, which unlike its amphotropic relativenormally only infects mouse cells, to specifically infect particularhuman cells. Replacement of a fragment of an envelope protein with anerythropoietin segement produced a recombinant retrovirus which thenbound specifically to human cells that expressed the erythropoietinreceptor on their surface, such as red blood cell precursors (Maulik andPatel 1997 “Molecular Biotechnology: Therapeutic Applications andStrategies” 1997. Wiley-Liss Inc. pp 45.).

[0097] In addition to gag, pot and env, the complex retroviruses alsocontain “accessory” genes which code for accessory or auxiliaryproteins. Accessory or auxiliary proteins are defined as those proteinsencoded by the accessory genes in addition to those encoded by the usualreplicative or structural genes, gag, pol and env. These accessoryproteins are distinct from those involved in the regulation of geneexpression, like those encoded by tat, rev, tax and rex. Examples ofaccessory genes include one or more of vif, vpr, vpx, vpu and nef. Theseaccessory genes can be found in, for example, HIV (see, for examplepages 802 and 803 of “Retroviruses” Ed. Coffin et al Pub. CSHL 1997).Non-essential accessory proteins may function in specialised cell types,providing functions that are at least in part duplicative of a functionprovided by a cellular protein. Typically, the accessory genes arelocated between pol and env, just downstream from env including the U3region of the LTR to or overlapping portions of the env and each other.

[0098] The complex retroviruses have evolved regulatory mechanisms thatemploy virally encoded transcriptional activators as well as cellulartranscriptional factors. These trans-acting viral proteins serve asactivators of RNA transcription directed by the LTRs. Thetranscriptional trans-activators of the lentiviruses are encoded by theviral tat genes. Tat binds to a stable, stem-loop, RNA secondarystructure, referred to as TAR, one function of which is to apparentlyoptimally position Tat to trans-activate transcription.

[0099] As mentioned earlier, retroviruses have been proposed as adelivery system (other wise expressed as a delivery vehicle or deliveryvector) for inter alia the transfer of a NOI, or a plurality of NOIs, toone or more sites of interest. The transfer can occur in vitro, ex vivo,in vivo, or combinations thereof. When used in this fashion, theretroviruses are typically called retroviral vectors or recombinantretroviral vectors. Retroviral vectors have even been exploited to studyvarious aspects of the retrovirus life cycle, including receptor usage,reverse transcription and RNA packaging (reviewed by Miller, 1992 CurrTop Microbiol Immunol 158:1-24).

[0100] In a typical recombinant retroviral vector for use in genetherapy, at least part of one or more of the gag, pot and env proteincoding regions may be removed from the virus. This makes the retroviralvector replication-defective. The removed portions may even be replacedby a NOI in order to generate a virus capable of integrating its genomeinto a host genome but wherein the modified viral genome is unable topropagate itself due to a lack of structural proteins. When integratedin the host genome, expression of the NOI occurs—resulting in, forexample, a therapeutic effect. Thus, the transfer of a NOI into a siteof interest is typically achieved by: integrating the NOI into therecombinant viral vector; packaging the modified viral vector into avirion coat; and allowing transduction of a site of interest—such as atargeted cell or a targeted cell population.

[0101] It is possible to propagate and isolate quantities of retroviralvectors (e.g. to prepare suitable titres of the retroviral vector) forsubsequent transduction of, for example, a site of interest by using acombination of a packaging or helper cell line and a recombinant vector.

[0102] In some instances, propagation and isolation may entail isolationof the retroviral gag, pol and env genes and their separate introductioninto a host cell to produce a “packaging cell line”. The packaging cellline produces the proteins required for packaging retroviral DNA but itcannot bring about encapsidation due to the lack of a psi region.However, when a recombinant vector carrying a NOI and a psi region isintroduced into the packaging cell line, the helper proteins can packagethe psi-positive recombinant vector to produce the recombinant virusstock. This can be used to infect cells to introduce the NOI into thegenome of the cells. The recombinant virus whose genome lacks all genesrequired to make viral proteins can infect only once and cannotpropagate. Hence, the NOI is introduced into the host cell genomewithout the generation of potentially harmful retrovirus. A summary ofthe available packaging lines is presented in “Retroviruses” (1997 ColdSpring Harbour Laboratory Press Eds: J M Coffin, S M Hughes, H E Varmuspp 449). However, this technique can be problematic in the sense thatthe titre levels are not always at a satisfactory level. Nevertheless,the design of retroviral packaging cell lines has evolved to address theproblem of inter alia the spontaneous production of helper virus thatwas frequently encountered with early designs. As recombination isgreatly facilitated by homology, reducing or eliminating homologybetween the genomes of the vector and the helper has reduced the problemof helper virus production.

[0103] More recently, packaging cells have been developed in which thegag, pol and env viral coding regions are carried on separate expressionplasmids that are independently transfected into a packaging cell lineso that three recombinant events are required for wild type viralproduction. This strategy is sometimes referred to as the three plasmidtransfection method (Soneoka et al 1995 Nucl. Acids Res. 23: 628-633).

[0104] Transient transfection can also be used to measure vectorproduction when vectors are being developed. In this regard, transienttransfection avoids the longer time required to generate stablevector-producing cell lines and is used if the vector or retroviralpackaging components are toxic to cells. Components typically used togenerate retroviral vectors include a plasmid encoding the Gag/Polproteins, a plasmid encoding the Env protein and a plasmid containing aNOI. Vector production involves transient transfection of one or more ofthese components into cells containing the other required components. Ifthe vector encodes toxic genes or genes that interfere with thereplication of the host cell, such as inhibitors of the cell cycle orgenes that induce apotosis, it may be difficult to generate stablevector-producing cell lines, but transient transfection can be used toproduce the vector before the cells die. Also, cell lines have beendeveloped using transient infection that produce vector titre levelsthat are comparable to the levels obtained from stable vector-producingcell lines (Pear et al 1993, PNAS 90:8392-8396).

[0105] In view of the toxicity of some HIV proteins—which can make itdifficult to generate stable HIV-based packaging cells—HIV vectors areusually made by transient transfection of vector and helper virus. Someworkers have even replaced the HIV Env protein with that of vesicularstomatis virus (VSV). Insertion of the Env protein of VSV facilitatesvector concentration as HIV/VSV-G vectors with titres of 5×10⁵ (10⁸after concentration) were generated by transient transfection (Naldiniet al 1996 Science 272: 263-267). Thus, transient transfection of HIVvectors may provide a useful strategy for the generation of high titrevectors (Yee et al 1994 PNAS. 91: 9564-9568).

[0106] If the retroviral component includes an env nucleotide sequence,then all or part of that sequence can be optionally replaced with all orpart of another env nucleotide sequence. Replacement of the env genewith a heterologous env gene is an example of a technique or strategycalled pseudotyping. Pseudotyping is not a new phenomenon and examplesmay be found in WO-A-98105759, WO-A-98/05754, WO-A-97/17457,WO-A-96/09400, WO-A-91100047 and Mebatsion et al 1997 Cell 90, 841-847.

[0107] Pseudotyping can confer one or more advantages. For example, withthe lentiviral vectors, the env gene product of the HIV based vectorswould restrict these vectors to infecting only cells that express aprotein called CD4. But if the env gene in these vectors has beensubstituted with env sequences from other RNA viruses, then they mayhave a broader infectious spectrum (Verma and Somia 1997 Nature389:239-242). By way of example—workers have pseudotyped an HIV basedvector with the glycoprotein from VSV (Verma and Somia 1997 ibid).Alternatively, env can be modified so as to affect (such as to alter)its specificity.

[0108] Thus, the term “recombinant retroviral vector” describes anentity (such as a DNA molecule) which contains sufficient retroviralsequences to allow an RNA transcript of the vector to be packaged in thepresence of essential retroviral proteins into a retroviral particlecapable of infecting a target cell. Infection of the target cellincludes reverse transcription and integration into the target cellgenome.

[0109] The term “recombinant retroviral vector” also covers a retroviralparticle containing an RNA genome encoded by the DNA molecule. Theretroviral vector will also contain non-viral genes which are to bedelivered by the vector to the target cell. A recombinant retroviralvector is incapable of independent replication to produce infectiousretroviral particles. Usually, a recombinant retroviral vector lacksfunctional gag-pol and/or env genes, or other genes encoding proteinsessential for replication.

[0110] The term “targeted retroviral vector” means a recombinantretroviral vector whose ability to infect a cell or to be expressed inthe target cell is restricted to certain cell types within the hostorganism. An example of targeted retroviral vectors is one with agenetically modified envelope protein which binds to cell surfacemolecules found only on a limited number of cell types in the hostorganism. Another example of a targeted retroviral vector is one whichcontains promoter and/or enhancer elements which permit expression ofone or more retroviral transcripts in only a proportion of the celltypes of the host organism.

[0111] Thus, the present invention provides a useful delivery system.The delivery system is capable of targeting an NOI and/or a POI to atumour—i.e. capable of homing a NOI and/or POI in on a tumour.

[0112] The vector may be used to administered directly to an entity—suchas an organism or a cell thereof—such as ex vivo or in vivo. In thissense the vector may be delivered directly, for example, to a tumoursite. Alternatively, the vector may be administered to an entity by wayof a carrier—such as ex vivo or in vivo. An example of a carrier wouldbe a liposome or a cell in which would be contained the vector. Anexample of a suitable carrier cell would be a haematpoietic cell, suchas a myeloid cell. These carrier cells may incease the furtherspecificity of the vector of the present invention.

[0113] These and other aspects of the present invention will now beelaborated on further.

[0114] The perceived potential of monoclonal antibody-based therapiesfor treatment of neoplastic disease has not been fully realised(reviewed in Scheinberg and Chapman 1995, In Monoclonal antibodies (ed.Birch and Lennox) Chapter 2.1; George et al., 1994 Immunol. Today 15;559-561). Consequently, monoclonal antibodies have been conjugated toradioisotopes, cytotoxic drugs or toxins in an attempt to improveefficacy. However, clinical trials with such conjugates have generallyled to disappointing results. One of the principal reasons for the lackof efficacy of antibodies and antibody conjugates in the treatment ofsolid tumours is the poor penetration of solid tumours byimmunoglobulins and other proteins such as immunotoxins of highmolecular weight (eg. Juweid et al. 1992, Cancer Res. 52; 5144-5153;Epenetos et al. 1986 Cancer Res. 46; 3183-319i). Other reasons for lackof efficacy include the non-specific toxicity, immunogenicity andinappropriate pharmacokinetics of many immunotoxins andantibody-radionuclide conjugates introduced into the systemiccirculation (reviewed in Scheinberg and Chapman 1995. In Monoclonalantibodies (ed. Birch and Lennox) Chapter 2.1).

[0115] In contrast to the general lack of in vivo efficacy, manymonoclonal antibodies show pronounced ability to inhibit the growth oftumour cells in certain in vitro assays (reviewed in Sandlie andMichaelsen 1996 In Antibody engineering: a practical approach. EdMcCafferty et al Chapter 9). It is well established that binding ofspecific antigen by an antibody can lead to activation of a variety ofeffector functions mediated via the Fc portion of the antibody heavychain. The Fc regions of different immunoglobulin classes mediatedifferent effector functions, including activation of complementcascades and binding to Fc receptors on various immune effector cells(Duncan et al 1988 Nature 332; 563 and 738). In in vitro assays,engagement of Fc receptors present on immune effector cells by antibodybound to tumour target cells can lead to destruction of the target cellby a variety of mechanisms collectively termed antibody dependentcellular cytotoxicity (ADCC). For example engagement of Fc-receptors forIgG, on human monocytes and macrophages, neutrophils and natural killer(NK) cells by antibodies of the IgG1 and IgG3 and to a much lesserextent IgG2 and IgG4 sub-classes, stimulates ADCC (Munn et al 1991Cancer Res. 51; 1117-1123; Primus et al., 1993 Cancer Res. 53;3355-3361). However, the relatively poor ability of such antibodies todestroy tumours in vivo suggests that ADCC does not play a significantrole in many of the current antibody—based therapies (George et al, 1994Immunol. Today 15; 559-561). There are several possible reasons forthis, including the poor penetration of antibodies into solid tumours(Yuan et al. 1995 Cancer Res. 55; 3752-3756) and the fact that themajority of the high-affinity receptor (FcgRI) molecules present onmacrophages are normally occupied by serum IgG which will be poorlycompeted by specific antibody (Munn et al 1991 Cancer Res. 51;1117-1123).

[0116] It has previously been shown that tumour cells transduced withgenes encoding monoclonal antibodies can participate in ADCC reactionsmediated by xenogeneic NK cells in vitro (Primus et al. 1993 Cancer Res.53: 3355-3361). However, NK cells play little role in the destruction oftumour cells in vivo, in part because their killing functions areinhibited by the presence of self MHC Class I on autologous tumour cells(Correa and Raulet 1995 Immunity 2; 61-71).

[0117] It has also been postulated that tumour infiltrating lymphocytes(TILs) could be used as a vehicle to deliver antibody genes to a tumourto secrete anti-tumour antibodies at the tumour site (Tsang et al 1993J. Immunother. 13; 143-152.) However, ex vivo transduction of TILsfollowed by autologous transplantation using marker genes has shown thatisolated TILs show no specific homing mechanism which could allow themto return to tumour deposits (Economou et al 1996 J. Clin. Invest. 97;515-521) and so any such approach is of limited value. The presentinvention is in contrast to these findings since there is provided avector that can target or deliver an NOI and/or a POI to a tumour mass(or site).

[0118] Transduction of a gene encoding a single-chain immunotoxin intohuman lymphokine—activated T-cells (LAK cells) has also been reported(Chen et al 1997 Nature 385, 78-80). In addition to the problems ofre-introducing the LAK cells to the site of the tumour, such an approachalso suffers from the potential drawbacks associated with beingrestricted to ex vivo use. These include the necessity of culturing theT-cells in high levels of a cytokine such as IL-2 to generate LAK cellswith consequent problems in generating sufficient cells for therapy.

[0119] In one aspect, the present invention relates to the use ofgenetic vectors to deliver genes (such as therapeutic genes) encodingsecreted tumour binding proteins (TBPs) to the interior of a tumour massand identifies ways to target expression of TBPs to the interior of thetumour. Expression of the gene or genes encoding the TBP within thetumour mass then leads to local production of TBP with consequentreduction of tumor growth, survival or dissemination by a variety ofmechanisms. Because the TBP is secreted, TBP produced by transducedcells can act not only on the transduced cell but on neighbouring tumourcells as well and hence achieve a bystander effect.

[0120] There are a number of cell types present within a tumor mass inaddition to the cancerous cells. These can include cells of the tumourvasculature (eg endothelial cells) and immune cells which infiltrate thetumour, such as tumour-infiltrating lymphocytes (TIL) and macrophages(Normann 1985 Cancer Metastasis Re. 4:277-291; Leek et al 1996 CancerRes. 56: 4625-4629). Any of these cell types can be targeted forexpression of the TBP and can serve as a local factory within the tumourfor production of TBP. Preferably, the cells in the tumour mass whichare used to produce the TBP are the cancerous cells, endothelial cellsor macrophages. Alternatively, the progenitors of monocytes orendothelial cells may be targeted, such as CD34-positive peripheralblood mononuclear cells (Asahara et al. 1997 Science 275: 964-967).

[0121] Preferably, the TBP comprises one or more binding domains capableof binding to one or more TACSMs which are present on the cancerouscells. Thus the TBP, produced from one or more of the cell types withinthe tumour mass is secreted and is directed to the cancerous cells byits affinity for the TACSM. The TACSM may be selectively present on arestricted number of cell types. Thus the amount of TACSM present on themajority of the cancerous cells within the tumour mass is higher than onsurrounding tissues. Preferably, the TACSM is detectably present only ontumour cells and a limited number of other tissue types in theindividual containing the tumour. More preferably, the TACSM isessentially tumour-specific in the individual containing the tumour.

[0122] The one or more binding domains of the TBP may consist of, forexample, a natural ligand for a TACSM, which natural ligand may be anadhesion molecule or a growth-factor receptor ligand (eg epidermalgrowth factor), or a fragment of a natural ligand which retains bindingaffinity for the TACSM. Alternatively, the binding domains may bederived from heavy and light chain sequences from an immunoglobulin (Ig)variable region. Such a variable region may be derived from a naturalhuman antibody or an antibody from another species such as a rodentantibody. Alternatively the variable region may be derived from anengineered antibody such as a humanised antibody or from a phage displaylibrary from an immunised or a non-immunised animal or a mutagenisedphage-display library. As a second alternative, the variable region maybe derived from a single-chain variable fragment (scFv). The TBP maycontain other sequences to achieve multimerisation or to act as spacersbetween the binding domains or which result from the insertion ofrestriction sites in the genes encoding the TBP, including Ig hingesequences or novel spacers and engineered linker sequences.

[0123] The TBP may comprise, in addition to one or more immunoglobulinvariable regions, all or part of an Ig heavy chain constant region andso may comprise a natural whole Ig, an engineered Ig, an engineeredIg-like molecule, a single-chain Ig or a single-chain Ig-like molecule.Alternatively, or in addition, the TBP may contain one or more domainsfrom another protein such as a toxin.

[0124] In one aspect of the invention, there is provided a gene deliverysystem for targeting one or more genes encoding a TBP to a tumour,comprising a genetic vector encoding a TBP and an in vivo gene-deliverysystem. The gene delivery system may be a non-viral gene delivery systemsuch as DNA compacted with a DNA-compaction agent, or a liposome orimmunoliposome which may contain DNA compacted with a DNA-compactionagent (such as a poly-lysine). The vector may be a plasmid DNA vector.Alternatively the vector may be a recombinant viral vector such as anadenovirus vector, an adeno-associated virus (AAV) vector, aherpes-virus vector or a retroviral vector in which case gene deliveryis mediated by viral infection of a target cell. Preferably the vectoris a recombinant retroviral vector, which may be a targeted retroviralvector. Preferably, the retroviral vector is resistant to humancomplement, for example by production in a human cell line.

[0125] Typically, the vector will contain a promoter to directexpression of the or each gene (such as a therapeutic gene) and maycontain additional genetic elements for the efficient or regulatedexpression of TBP genes, including enhancers, translation initiationsignals, internal ribosome entry sites (IRES), splicing andpolyadenylation signals. The promoter and/or enhancer may betissue-restricted in its activity. For example a tumour-specificpromoter-enhancer, such as a 5T4 antigen gene promoter-enhancer or theCEA-gene promoter-enhancer may be used. Alternatively, or additionally,an element or elements for regulated expression may be present, such asa hypoxia regulated enhancer. An example of a hypoxia regulatedexpression element (HRE) is a binding element for the transcriptionfactor HIF1. The enhancer elements or elements conferring regulatedexpression may be present in multiple copies. Preferably, expression ofthe or a gene (such as a therapeutic gene) is inducible by hypoxia (orlow oxygen supply) such as may be found in a tumour mass. Mostpreferably, the promoter and/or enhancer directing expression of thegene (such as a therapeutic gene) contains both hypoxia-responsiveelements and elements which give higher expression in tumour cells thanin neighbouring non-tumour cells. Additional vector components will beprovided for other aspects of vector function such as vectormaintenance, nuclear localisation, replication, and integration asappropriate using components which are well known in the art.

[0126] In a preferred embodiment of this aspect of the invention, aretroviral vector is provided for in vivo delivery of the gene or genesencoding the TBP to the tumour. Suitable retroviral vectors are known inthe art (see for example Gunzberg and Salmons 1996 In Gene Therapy ed.Lemoine and Cooper. Bios; and Cosset et al. 1995 J. Virol. 69;7430-7436). In a particularly preferred embodiment, expression of theTBP may be enhanced in the hypoxic regions of the tumour by theinclusion of hypoxia regulated genetic elements in the retroviralvector. In this case, the hypoxia-regulated elements may be insertedinto one or both of the retroviral LTRs in place of the LTR enhancer orin another position in the vector, by standard molecular biologytechniques. The gene or genes encoding the TBP may be expressed from apromoter-enhancer which leads to enhanced expression in the tumour cellscompared with neighbouring non-tumour cells or is preferably essentiallytumour-specific. Examples of suitable promoters include thepromoter-enhancer of the gene for 5T4 antigen, the promoter-enhancer ofthe MUC1 gene or the CEA gene.

[0127] In an other aspect of the invention there is provided a method oftreating cancer comprising administering the TBP gene or genes in a genedelivery system of the first aspect of the invention either systemicallyor directly to the site of a tumour.

[0128] In an other aspect of the invention, is provided a gene deliverysystem for introducing one or more genes encoding a TBP into cells ofthe haematopoietic (preferably myeloid haematopoietic) cell lineageeither in vivo or ex vivo. Preferably the haematopoietic (preferablymyeloid haematopoietic) cells are of the monocyte-macrophage lineage ora precursor of such cells such as a CD34-positive stem cell. For ex vivodelivery, the genes can be inserted into a plasmid vector and deliveredby one of a variety of DNA transfection methods includingelectroporation, DNA biolistics, lipid-mediated transfection orcompacted DNA-mediated transfection. Alternatively a viral vector can beused to transduce haematopoietic (preferably myeloid haematopoietic)cells or CD34-positive stem cells ex vivo, such as an adenovirus vector,a retroviral vector or a lentiviral vector. The vector will contain apromoter to direct expression of the or each gene (such as a therapeuticgene) and may contain additional genetic elements for efficient orregulated expression including enhancers, translation initiationsignals, internal ribosome entry sites (IRES), splicing andpolyadenylation signals. The promoter, or an enhancer or splicingsignals may be tissue-restricted and preferentially active inmononuclear phagocytes such as macrophages. The promoter and/or enhancermay contain elements for regulated expression such as ahypoxia-regulated enhancer. An example of a hypoxia regulated expressionelement is HIF1 transcription factor response element. Such an elementmay be present in multiple copies. Examples of hypoxia-regulatedpromoters and enhancers include those from the enolase gene, theerythropoietin gene, and genes encoding glycolytic enzymes (Semenza etal., 1994 J. Biol. Chem 269; 23757-23763) such as the PGK gene. IsolatedHREs can be multimerised in order to increase the response to hypoxia.Additional vector components may be provided for other aspects of vectorfunction such as vector maintenance, nuclear localisation, replicationand integration as appropriate using components which are well known inthe art.

[0129] After introduction of the vector into the cells ex vivo, thecells can be re-introduced into the patient directly or they can bestimulated to differentiate along the monocyte-macrophagedifferentiation pathway using appropriate combinations of cytokines andgrowth factors prior to re-introduction into the patient. CD34-positivecells are stimulated to differentiate using cytokines including IL-3,GMCSF and MCSF. Monocytes are differentiated either by culture attachedto plastic or using GMCSF either alone or in combination with othercytokines including MCSF.

[0130] For introduction of genes (such as therapeutic genes) intohaematopoietic (preferably myeloid haematopoietic) cells orCD34-positive stem cells in vivo, a suitable in vivo delivery system canbe used to deliver the transcription units described above. The genedelivery system may be a non-viral gene delivery system such as DNAcompacted with a DNA-compaction agent, or a liposome or immunoliposomewhich may contain DNA compacted with a DNA-compaction agent.Alternatively the vector may be a recombinant viral vector such as atargeted adenovirus vector, an adeno-associated viral (AAV) vector, aherpes-virus vector or a retroviral vector such as a lentiviral vector.Preferably the vector is a targeted recombinant retroviral vector, whichis preferably resistant to human complement, for example by preparationof the vector from a human packaging cell line.

[0131] CD34-positive stem cells can also differentiate to formendothelial cells (Ashara et al. 1997 Science 275; 964-967). Such aroute of differentiation for CD34 positive stem cells containing TBPencoding genes according to the invention is envisaged in addition todifferentiation to form monocytes and macrophages.

[0132] Additional vector components may be provided for other aspects ofvector function such as vector maintenance, nuclear localisation,replication, and integration as appropriate using components which arewell known in the art.

[0133] In a preferred embodiment of this aspect of the invention, aplasmid vector or a retroviral vector carrying a gene encoding a TBPunder the control of a hypoxia regulated promoter or a promoterpreferentially active in macrophages is introduced into autologousperipheral blood monocytes. The transfected monocytes are re-introducedinto the patient where they migrate to the hypoxic regions of tumourspermitting enhanced production of the TBP in the interior of the tumourmass. The macrophages are optionally treated with cytokines prior tore-injection into the patient. Alternatively or additionally the vectormay include DNA sequences capable of expressing a cytokine gene such asa gene for IFNg, CSF-1 or GM-CSF in order to elicit the differentiationof the transfected cells. The cytokine gene may also be regulated bygenetic elements which show enhanced activity at the site of the tumour.

[0134] In an other aspect of the invention, there is provided a methodfor treating cancer in a human or non-human mammal, comprisingwithdrawing an amount of blood from an individual suffering from cancer,preparing from the blood a cell preparation enriched in monocytes andmacrophages or their stem-cell progenitors, introducing TBP genes intothe cell preparation using a gene delivery system of the third aspect ofthe invention so as to bring about transfection or transduction of themonocytes and macrophages, or their stem-cell progenitors with the TBPgenes, and re-introducing the transfected or transduced cells eithersystemically or directly to the site of the tumour. The cellpreparations may optionally be treated with cytokines prior toreintroduction in order to elicit differentiation towards activemacrophages.

[0135] In an other aspect of the invention is provided a method fortreating cancer in a mammal, comprising administering to an individual agene delivery system of the invention capable of expressing a TBP incells derived from a haematopoietic (preferably myeloid haematopoietic)origin.

[0136] In a further aspect of the invention there is provided a geneticvector comprising a gene (such a therapeutic gene) or genes encoding aTBP, operably linked to an expression regulatory element selectivelyfunctional in a cell type present within a tumour mass. The TBP in thisaspect of the invention inhibits tumour function by binding to a TACSMhaving an essential role in tumour cell survival or dissemination. TheTACSM in this aspect of the invention may be a cell surface moleculewhich has a role in tumour cell growth, migration or metastasis, and ispresent on cancerous cells or on another cell type within the tumourmass. Preferably the TACSM is present on cancerous cells or tumourvasculature or on macrophages and is a molecule such as a growth-factorreceptor, a plasminogen activator, a metalloproteinase or the 5T4antigen. The gene or genes encoding the TBP may be delivered to theinterior of the tumour by any of the routes described in the above twoaspects of the invention. Binding of the TBP to the corresponding TACSMblocks the function of the TACSM and thereby leads to inhibition ofgrowth, migration or metastasis of the tumour.

[0137] In a yet further aspect of the invention, a genetic vectorcomprising a gene (such as a therapeutic gene or genes) is delivered tothe interior of the tumour wherein the gene (such as a therapeutic gene)encodes a TBP, which additionally contains one or more effector domains.The effector domain or domains may be activated on binding of the TBP toa TACSM leading to inhibition of tumour cell proliferation, survival ordissemination. The TACSM in this aspect of the invention is a cellsurface molecule for which a specific TBP is available such as a tumourspecific carbohydrate moiety, an oncofoetal antigen, a mucin, agrowth-factor receptor or another glycoprotein. The TACSM is preferablyan antigen restricted in its tissue distribution and found predominantlyon the tumour cells and on the majority of cells within the tumour.Alternatively, the TACSM is present on tumour macrophages or the tumourvasculature. In some instances, the TACSM is not shed from the cellsurface into the circulation to an appreciable extent. However, sheddingmay occur. By way of example, shedding of the 5T4 antigen into thestroma can serve to further localise the NOI and/or the POI to thetumour environment.

[0138] The effector domain of the present invention may possessenzymatic activity and may be for example a pro-drug activating enzyme,or it may be a non-enzyme domain. Examples of TBPs containing effectordomains with enzyme activity include antibody—enzyme conjugates orfusions. Antibody—enzyme conjugates have been described includingconjugates with alkaline phosphatase (Senter et al., 1988 Proc. Natl.Acad. Sci. 85: 4842-4846); carboxypeptidase G2 (Bagshawe et al. 1988 Br.J. Cancer 58: 700703); P-lactamase (Shepherd et al 1991 Bioorg. Med.Chem. Left. 1:21-26); and Penicillin-V-amidase (Kerr et al. 1990 CancerImmunol. Immunother. 31: 202-206. Antibody—enzyme fusions have also beendescribed (Goshorn et al 1993 Cancer Res 53: 2123-2127; Wels et al 1992Bio/Technology 10: 1128-1132). Each of these examples can be used inthis aspect of the invention. Additional or alternative enzymes whichmay be included in TBPenzyme fusions include human Carboxypeptidase Alor a mutant thereof (Smith et al 1997 J. Biol. Chem. 272: 15804-15816);cytosine deaminase (Mullen et al. 1994 Cancer Res. 54: 1503-1506); HSVthymidine kinase (Borrelli et al. 1988 Proc. Natl. Acad. Sci. 85:7572-7576.); nitroreductase; P450-Reductase and a P450.

[0139] Preferably the pro-drug activating enzyme domain or domains aregenetically fused to the C-terminus of an immunoglobulin orimmunoglobulin domain such as a scfv or a single-chain antibody orFab-fragment. In a particularly preferred embodiment of this aspect ofthe invention, the immunoglobulin domain or domains are human orhumanised and the enzyme is a human enzymp—such as a Carboxypeptidase aP450 or P450-Reductase. The enzyme may be a mutant enzyme which convertsa pro-drug more efficiently than does the native human enzyme. Inaccordance with the present invention, any enzyme that has utility in anADEPT strategy can be used.

[0140] In each case, a suitable pro-drug is used in the treatment of thepatient in combination with the appropriate pro-drug activating enzyme.Examples of pro-drugs include etoposide phosphate (used with alkalinephosphatase Senter et al., 1988 Proc. Nat. Acad. Sci. 85: 4842-4846);5-fluorocytosine (with Cytosine deaminase Mullen et al 1994 Cancer Res.54: 1503-1506); Doxorubicin-N-p-hydroxyphenoxyacetamide (withPenicillin-V-Amidase (Kerr et al. 1990 Cancer Immunol. Immunother. 31:202-206); Para-N-bis(2chloroethyl) aminobenzoyl glutamate (withCarboxypeptidase G2); Cephalosporin nitrogen mustard carbamates (withP-lactamase); SR4233 (with P450 Reducase); Ganciclovir (with HSVthymidine kinase, Borrelli et al. 1988 Proc. Natl. Acad. Sci. 85:7572-7576) mustard pro-drugs with nitroreductase (Friedlos et al. 1997 JMed Chem 40: 1270-1275) and Cyclophosphamide (with P450 Chen et al. 1996Cancer Res 56: 1331-1340).

[0141] Alternatively the effector domain may be a non-enzyme domain.Examples of non-enzyme effector domains include toxins such an exotoxinfrom a pseudomonad bacterium, all or part of a cytokine such as IL-2 orIFNγ, or effector domains from immunoglobulin heavy chains.

[0142] In a preferred embodiment of this aspect of the invention, theTBP contains an effector domain capable of activating macrophage FcgR I,II or III receptors. On binding of the TBP to antigen on the tumourcells, macrophages present within the hypoxic regions of the tumour areactivated to destroy the tumour cells directly by phagocytosis or ADCCor are activated to secrete pro-inflammatory cytokines which serve toenhance the natural immunological response to the tumour. The TBP maycontain an Fc region from an immunoglobulin, a mutant Fc region, areceptor-binding fragment of the Fc region or may contain anotherFcR—binding domain.

[0143] Preferably the TBP contains an entity, preferably an effectordomain entity, that confers protein stability ex vivo and/or in vivo.

[0144] In accordance with the present invention, the TBP may include anintact Fc region from an IgG, (such as human IgG1 or IgG3), preferablyfrom IgE (such as human IgGE), or a part thereof.

[0145] In one preferred embodiment of this aspect of the invention, theTBP is a Sab (single chain antibody) containing a human IgG1 constantregion and a binding domain which recognises the 5T4 antigen.

[0146] In a particularly preferred embodiment of this aspect of theinvention, the TBP is a Sab (single chain antibody) containing a humanIgE constant region and a binding domain which recognises the 5T4antigen.

[0147] The effector domain may be encoded by a portion of a cDNA fusedin-frame to the DNA encoding the tumour-binding domain. Alternatively agenomic fragment containing introns may be used such as a human IgG1heavy chain constant region genomic fragment.

[0148] Here the term “intron” is used in its normal sense—e.g. anintervening sequence of DNA within a gene which is removed by RNAsplicing and so is not present in the mature messenger RNA and does notcode for protein. Introns can be conditional or alternatively spliced indifferent cell types.

[0149] Introduction of TBP-encoding genes into monocytes or macrophagesmay be combined with further treatments to elicit macrophagedifferentiation and activation. For example, cells maintained ex vivomay be treated with cytokines such as IFNγ, CSF-1 or GM-CSF prior tore-introduction into the patient. Alternatively, genes encoding thesecytokines may be introduced into the monocytes/macrophages in the sameor a different vector from the TBP genes in vivo or ex vivo.Consequently in a still further aspect of the invention there isprovided a method of treating cancer in a mammal which comprisesadministering to an individual a combination of a cytokine or acytokine-encoding gene and one or more TBP genes according to any of theprevious aspects of the invention.

[0150] In accordance with the invention, standard molecular biologytechniques may be used which are within the level of skill in the art.Such techniques are fully described in the literature. See for example;Sambrook et al. (1989) Molecular Cloning; a laboratory manual; Hames andGlover (1985-1997) DNA Cloning: a practical approach, Volumes I-IV(second edition). Methods for the engineering of immunoglobulin genes inparticular are given in McCafferty et al (1996) Antibody engineering: apractical approach.

[0151] In a preferred aspect, the present invention relates to thedelivery of TBP-encoding genes to the site of a tumour. This hasconsiderable advantages for medical applications (such as therapeuticapplications) in which TBPs are indicated since it circumvents a numberof problems associated with delivery of proteins systemically in humans.

[0152] In contrast to the problems associated with production anddelivery of proteins, the methods of the invention allow the delivery ofgenes to the site of the tumour, thus circumventing a number ofproduction problems. The TBPs are thereby produced in situ in theautologous human cells, which serve as a local factory for theproduction of the gene-based medicament (such as a therapeutic). Thishas significant advantages in minimising systemic toxicity. The activityof the protein is maximal since the glycosylation of the protein shows ahuman pattern appropriate to the individual being treated.

[0153] The methods of the invention can be used in conjunction withdirect injection into the site of the tumour or systemic delivery of,for example targeted vectors or engineered haematopoietic (preferablymyeloid haematopoietic) cells or their progenitors. Systemic deliverymay be particularly advantageous in a number of indications,particularly in the treatment. of disseminated disease. In these casesthe gene delivery system or engineered cells can be administeredintravenously by bolus injection or by infusion in a suitableformulation. A pharmaceutically acceptable formulation may include anisotonic saline solution, a buffered saline solution or a tissue-culturemedium. Additional formulatory agents may be included such aspreservative or stabilising agents.

[0154] Thus, the present invention also encompasses a pharmaceuticalcomposition for treating one or more individuals by gene therapy,wherein the composition comprises a therapeutically effective amount ofthe vector according to the present invention or the expressed productthereof. The pharmaceutical composition may be for human or animalusage. Typically, a physician will determine the actual dosage whichwill be most suitable for an individual subject and it will vary withthe age, weight and response of the particular patient.

[0155] The composition may optionally comprise a pharmaceuticallyacceptable carrier, diluent, excipient or adjuvant. The choice ofpharmaceutical carrier, excipient or diluent can be selected with regardto the intended route of administration and standard pharmaceuticalpractice. The pharmaceutical compositions may comprise as—or in additionto—the carrier, excipient or diluent any suitable binder(s),lubricant(s), suspending agent(s), coating agent(s), solubilisingagent(s), and other carrier agents that may aid or increase the viralentry into the target site (such as for example a lipid deliverysystem).

[0156] Where appropriate, the pharmaceutical compositions can beadministered by any one or more of: inhalation, in the form of asuppository or pessary, topically in the form of a lotion, solution,cream, ointment or dusting powder, by use of a skin patch, orally in theform of tablets containing excipients such as starch or lactose, or incapsules or ovules either alone or in admixture with excipients, or inthe form of elixirs, solutions or suspensions containing flavouring orcolouring agents, or they can be injected parenterally, for exampleintracavernosally, intravenously, intramuscularly or subcutaneously. Forparenteral administration, the compositions may be best used in the formof a sterile aqueous solution which may contain other substances, forexample enough salts or monosaccharides to make the solution isotonicwith blood. For buccal or sublingual administration the compositions maybe administered in the form of tablets or lozenges which can beformulated in a conventional manner.

[0157] Thus, a preferred aspect of the present invention relates to avector comprising (a) a NS coding for a TIP and (b) an NOI which encodesa POI; wherein the TIP is capable of recognising a tumour, such that inuse the vector is capable of delivering the NOI and/or the POI to thetumour.

[0158] In one exemplary embodiment of the present invention TIP is IgGor IgE or a part thereof.

[0159] In another exemplary embodiment of the present invention, TIP isEGF or a part thereof.

[0160] In another exemplary embodiment of the present invention, TIPrecognises a trophoblast cell surface antigen and at least one of theeffector domains is a secreted co-stimulatory molecule. Furtherbackground teaching and details on this embodiment now follow.

[0161] The latter-mentioned aspect of the present invention relates to aprocess for the activation of lymphocytes and the use of activatedlymphocytes in the treatment of cancer. It also relates to fusionproteins for the activation of lymphocytes, to nucleic acids encodingthe fusion proteins and to vectors carrying the nucleic acids.

[0162] Lymphocytes require at least two distinct signals in order torespond to antigens by activation of effector functions (Bretscher andCohn 1970 Science 169: 1042-1049; Crabtree 1989 Science 243: 355-361).The primary signal is specific for antigen. For B-lymphocytes, theB-cell antigen receptor (surface immunoglobulin) recognisesthree-dimensional epitopes on a variety of macromolecules. ForT-lymphocytes, the T-cell receptor (TCR) recognises peptide antigensdisplayed on the surface of antigen-presenting cells by proteins of themajor histocompatability (MHC) family (Weiss et al. 1986 Ann. Rev.Immunol. 4: 593-619).

[0163] Stimulation of the primary signal in isolation normally leads toapoptosis (programmed cell death) of the lymphocyte or leads to theestablishment of a state of sustained unresponsiveness or anergy (Weisset al. supra). In order to achieve activation of the lymphocyte,accessory signals are required which may be delivered by cytokines or bycell-surface co-stimulatory ligands present on antigen-presenting cells(APC).

[0164] There are a number of such co-stimulatory molecules nowidentified including adhesion molecules, LFA-3, ICAM-1, ICAM-2. Majorco-stimulatory molecules present on APC are the members of the B7 familyincluding B7-1 (CD80), B7-2 (CD86) and B7-3. These molecules are ligandsof co-stimulatory receptors on lymphocytes including CD28 (WO92/00092),probably the most significant co-stimulatory receptor for restingT-cells. Different members of the B7 family of glycoproteins may deliversubtly different signals to T-cells (Nunes et al. 1996 J. Biol. Chem.271: 1591-1598).

[0165] Established tumours, despite the fact that they commonly expressunusual antigens on their surfaces, are poorly immunogenic. It has beenpostulated previously that one method for stimulating immune recognitionof tumour cells would be to enhance antigen presentation andco-stimulation of lymphocytes in the context of tumour antigens.Transfection of the genes encoding B7-1 and B7-2, alone or incombination with cytokines, have been shown to enhance the developmentof immunity to experimental tumours in animal models (e.g. Leong et al.1997 Int. J. Cancer 71: 476-482; Zitvogel et al. 1996 Eur. J. Immunol.26:1335-1341; Cayeux et al. 1997 J. Immunol 158:2834-2841). However, intranslating these results into a practical treatment for human cancer,there are a number of significant problems to be overcome. A majorproblem in such studies is the need to deliver B7 genes in vivo to alarge number of cells of the tumour to achieve efficacy. A secondproblem is that it is important to target expression of B7 to the tumourcells to avoid inappropriate immune cell activation directed againstother cell types.

[0166] This aspect of the present invention solves these specificproblems by delivering a gene encoding a secreted co-stimulatorymolecule (“SCM”) with binding affinity for a tumour antigen. In thisway, a relatively small number of transfected cells within the tumouract as a local factory to produce the co-stimulatory molecule which isshed from the producer cell and binds to other cells in the tumour. Theaspect of the present invention has the additional advantage that tumourcells need not be the target for transfection.

[0167] The SCM of the invention is a novel engineered fusion proteincomprising a signal peptide for secretion from mammalian cells, at leastone antigen-binding domain from an immunoglobulin or animmunoglobulin-like molecule and at least one further domain which actsas a co-stimulatory signal to a cell of the immune system. The use ofcombinations of SCMs containing different co-stimulatory domains is alsoenvisaged. The SCMs are produced by expression of SCM-encoding genes inthe autologous cells of the individual to be treated and hence anypost-translational modifications added to the protein by the host cellare authentic and provide fully functional protein and appropriatepharmacokinetics.

[0168] WO-A-92/00092 describes truncated forms of B7-1, derived byplacing a translation stop codon before the transmembrane domain,secreted from mammalian cells. In that particular case, a heterologoussignal peptide from the Oncostatin M gene was used. WO-A92/00092 alsodescribes fusion proteins which contain the extracellular domain of B7-1fused to the Fc region of an immunoglobulin. Such molecules can bind toCD28 on T-cells and serve to stimulate T-cell proliferation. Howeversuch stimulation occurs only to a moderate extent unless the B7 orB7-derivative is immobilised on a solid surface.

[0169] Gerstmayer et al. (1997 J. Immol. 158: 4584-4590) describes afusion of B7-2 to an scFv specific for ErbB2 followed by a myc epitopetag and polyhistidine tag which is secreted when expressed in the yeastPichia pastoris. This molecule retained binding for antigen andco-stimulated proliferation of T-cells prestimulated with PMA and IL-2.However, glycosylation of such a molecule is of the yeast type, which islikely to lead to inappropriate pharmacokinetics in humans.

[0170] In accordance with the present invention, any suitableco-stimulalatory domain(s) may be used. By way of example,co-stimulatory domains can be chosen from extracellular portions of theB7 family of cell-surface glycoproteins, including B7-1, B7-2 and B7-3or other co-stimulatory cell surface glycoproteins such as but notlimited to co-stimulatory receptor-ligand molecules including CD2/LFA-3,LFA-1/ICAM-1 and ICAM-3. Studies have demonstrated that T cellco-stimulation by monocytes is dependent on each of two receptor ligandpathways CD2/LFA-3 and LFA-1/ICAM-1 (Van Seventer et al 1991 Eur JImmunol 21: 1711-1718). In addition, it has been shown that ICAM-3, thethird LFA-1 counterreceptor, is a co-stimulatory molecule for restingand activated T lymphocytes (Hernandez-Caselles et at 1993 Eur J Immunol23: 2799-2806).

[0171] Other possible co-stimulatory molecules may include a novelglycoprotein receptor designated SLAM, has been identified which, whenengaged, potentiates T-cell expansion in a CD28-independent manner andinduces a Th0/Th1 cytokine production profile (Cocks et al 1995 Nature376: 260-263).

[0172] CD6, a cell surface glycoprotein, has also been shown to functionas a co-stimulatory and adhesion receptor on T cells. Four CD6 isoforms(CD6a, b, c, d) have been described (Kobarg et at 1997 Eur J Immunol 27:2971-2980). A role for the very late antigen (VLA-4) integrin in theactivation of human memory B cells has also been suggested (Silvy et al1997 Eur J Immunol 27: 2757-2764). Endothelial cells also provide uniqueco-stimulatory signals that affect the phenotype of activated CD4+ Tcells (Karmann et al 1996 Eur J Immunol 26: 610-617). A B3 protein,present on the surface of lipopolysaccharide-activated B cells, whichcan provide co-stimulation to resting T cells leading to a predominantrelease of interleukin (IL)-4 and IL-5 and negligible amounts of IL-2and interferon gamma has been described (Vinay et at 1995 J Biol Chem270: 23429-23436). The co-expression of a novel co-stimulatory T cellantigen (A6H) on T cells and tumour cells has suggested a possiblefunction related to common properties of these cells (Labuda et al 1995Int Immunol 7: 1425-1432).

[0173] In one preferred embodiment of the invention, the co-stimulatorydomain is a portion of B7-1 or B7-2, more preferably the completeextracellular portion of B7-1 or B7-2.

[0174] The SCM is formed by expression of a novel gene encoding a fusionprotein containing the antigen-binding domain or domains and theco-stimulatory domain or domains. If the antigen-binding domain iscomprised of a heavy and a light chain, the co-stimulatory domain isfused to one or other of the immunoglobulin chains, preferably to theheavy chain. If the antigen-binding domain is a scFv, the co-stimulatorydomain is fused to the scFv. The domains can be placed in the order(N-terminus to C-terminus): antigen-binding domain followed byco-stimulatory domain; or co-stimulatory domain followed byantigen-binding domain. Preferably, the co-stimulatory domain is placedat the N-terminus followed by the antigen-binding domain. A signalpeptide is included at the N-terminus, and may be for example thenatural signal peptide of the co-stimulatory extracellular domain. Thedifferent domains may be separated by additional sequences, which mayresult from the inclusion of convenient restriction-enzyme cleavagesites in the novel gene to facilitate its construction, or serve as apeptide spacer between the domains, or serve as a flexible peptidelinker or provide another function. Preferably the domains are separatedby a flexible linker.

[0175] Two or more different genes encoding different SCMs may be usedto achieve improved co-stimulation, or both co-stimulation of naiveT-cells and induction of memory responses. For example a gene encodingan SCM containing the B7-1 extracellular domain may be administered witha gene encoding an SCM containing the B7-2 extracellular domain.

[0176] Thus in one aspect of the invention, there is provided one ormore genetic vectors capable of expressing in mammalian cells one ormore secreted co-stimulatory molecules, each secreted co-stimulatorymolecule comprising at least one antigen-binding domain and at least onedomain from the extracellular portion of a cell-surface co-stimulatorymolecule. The co-stimulatory domain may be obtained from a moleculeexpressed on the surface of an antigen-presenting cell such as a B7family member. Preferably the co-stimulatory domain is from B7-1, B7-2or B7-3. Most preferably it is comprised of B7-1 amino acid residues 1to approximately 215 of the mature B7-1 molecule (described inWO-A-96/00092) or amino acids 1 to approximately 225 of the maturecell-surface form of B7-2 (described in Gerstmeyer et al. 1997 J.Immunol. 158:4584-4590).

[0177] The genetic vector according to this aspect of the inventioncomprises at least a promoter and enhancer for expression in mammaliancells and a polyadenylation site. Suitable promoters and enhancersinclude the MIE promoter-enhancer from human cytomegalovirus orpromoters which are expressed preferentially in cells present within thetumour. Such promoter-enhancers include those from the MUC1 gene, theCEA gene or the 5T4antigen gene. If two or more SCMs are expressed, thecoding regions for these may be inserted into two separate vectors or asingle vector may be used to express the two or more genes. In thelatter case each gene is provided with a separate copy of the promoter,or an internal ribosome entry site (IRES) is used to separate the twocoding sequences.

[0178] The present invention also covers the use of mutants, variants,homologues or fragments of the sequences disclosed herein

[0179] The terms “variant”, “homologue” or “fragment” in relation to thenucleotide sequences include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) nucleic acid from or to the sequence providing the resultantnucleotide sequence codes for or is capable of coding for an entityhaving the same function as that presented herein, preferably being atleast as biologically active as the same. In particular, the term“homologue” covers homology with respect to structure and/or functionproviding the resultant nucleotide sequence codes for or is capable ofcoding for an entity having the same function as that presented herein.With respect to sequence homology, preferably there is at least 75%,more preferably at least 85%, more preferably at least 90% homology tothe sequences shown herein. More preferably there is at least 95%, morepreferably at least 98%, homology to the sequences shown herein.

[0180] In particular, the term “homology” as used herein may be equatedwith the term “identity”. Relative sequence homology (i.e. sequenceidentity) can be determined by commercially available computer programsthat can calculate % homology between two or more sequences. A typicalexample of such a computer program is CLUSTAL.

[0181] The terms “variant”, “homologue” or “fragment” are synonymouswith allelic variations of the sequences.

[0182] The term “variant” also encompasses sequences that arecomplementary to sequences that are capable of hybridising to thenucleotide sequences presented herein. Preferably, the term “variant”encompasses sequences that are complementary to sequences that arecapable of hybridising under stringent conditions (e.g. 65° C. and0.1×SSC {1×SSC=0.15 M NaCl, 0.015 Na₃ citrate pH 7.0}) to the nucleotidesequence presented herein.

[0183] The present invention also covers nucleotide sequences that canhybridise to the nucleotide sequences of the present invention(including complementary sequences of those presented herein). In apreferred aspect, the present invention covers nucleotide sequences thatcan hybridise to the nucleotide sequence of the present invention understringent conditions (e.g. 65° C. and 0.1×SSC) to the nucleotidesequence presented herein (including complementary sequences of thosepresented herein).

[0184] The terms “variant”, “homologue” or “fragment” in relation to theamino acid sequences include any substitution of, variation of,modification of, replacement of, deletion of or addition of one (ormore) amino acid from or to the sequence providing the resultant aminoacid sequence has the same function as that presented herein, preferablybeing at least as biologically active as the same. In particular, theterm “homologue” covers homology with respect to structure and/orfunction providing the resultant amino acid sequence has the samefunction as that presented herein. With respect to sequence homology,preferably there is at least 75%, more preferably at least 85%, morepreferably at least 90% homology to the sequences shown herein. Morepreferably there is at least 95%, more preferably at least 98%, homologyto the sequences shown herein.

[0185] In summation, the present invention relates to a vectorcomprising (a) a NS coding for a TIP and optionally (b) an NOI whichencodes a POI; wherein the TIP is capable of recognising a tumour, suchthat in use the vector is capable of delivering the NOI and/or the POIto the tumour.

[0186] A preferred aspect of the present invention relates to a vectorcomprising (a) a NS coding for a TIP and (b) an NOI which encodes a POI;wherein the TIP is capable of recognising a tumour, such that in use thevector is capable of delivering the NOI and/or the POI to the tumour.

[0187] A further preferred aspect of the present invention relates to avector comprising (a) a NS coding for a TIP and (b) an NOI which encodesa POI; wherein the TIP is capable of recognising a tumour, such that inuse the vector is capable of delivering the NOI and/or the POI to thetumour; and wherein the TIP and POI are fused to each other.

[0188] This aspect of the present invention is advantageous as it allowsfor the production and delivery of, for example, a fusion product thatcomprises an effector component and a targetting component.

[0189] A further preferred aspect of the present invention relates to avector comprising (a) a NS coding for a TIP and (b) an NOI which encodesa POI; wherein the TIP is capable of recognising a tumour, such that inuse the vector is capable of delivering the NOI and/or the POI to thetumour; wherein the TIP and POI are fused to each other; and wherein thePOI is capable of being secreted.

[0190] This aspect of the present invention is highly advantageous as itprovides a means for the in situ production of a POI by, for example, asmall number of cells for the subsequent delivery of at least a portionof the produced POI to at least one neighbouring cell. Thus, one needonly infect a small number of cells to achieve a beneficial therapeuticeffect.

[0191] Thus, alternatively expressed, the present invention provides theuse of a vector according to the present invention as an in situproduction factory of any one or more of the NS, NOI, POI and TIP.

[0192] In addition, the present invention provides the use of a vectoraccording to the present invention when present in a cell to deliver anNOI and/or POI to a neighbouring cell.

[0193] A more preferred aspect of the present invention relates to avector comprising (a) a NS coding for a TIP, (b) an NOI which encodes aPOI, and (c) a nucleotide sequence that codes for a secretory entity;wherein the TIP is capable of recognising a tumour, such that in use thevector is capable of delivering the NOI and/or the POI to the tumour;wherein the TIP and POI are fused to each other; and wherein the POI iscapable of being secreted.

[0194] The invention will now be further described by way of examples,which are meant to serve to assist one of ordinary skill in the art incarrying out the invention and are not intended in any way to limit thescope of the invention. Reference is made to the following Figures:

[0195]FIG. 1a—which shows a DNA sequence encoding a 5T4 scFv, designated5T4scFv.1. The sequence of the mature secreted protein is given.

[0196]FIG. 1b—which shows the cDNA sequence encoding 5T4Sab1. Thesequence begins with a HindIII restriction site followed by atranslation initiation signal and a signal peptide.

[0197]FIG. 2 shows the sequence of B7-1.5T4.1

[0198]FIG. 3 shows a diagrammatic representation of two SCMs based onthe B7-1 co-stimulatory domain; FIG. 3a shows the SCM B7-1.5T4.1 andFIG. 3b shows B7-1.5T4.2 in which the order of the co-stimulatory andtumour-binding domains are reversed. Sp=signal peptide; B7ec=extracellular domain of B7-1; Vl=light chain variable domain of 5T4;Vh=heavy chain variable domain of 5T4.

[0199]FIG. 4 shows the sequence of the extracellular domain of humanB7-2, including the signal peptide sequence. The mature protein beginsat amino acid 17. The B7-2 derived sequence is followed by a flexiblelinker gly-gly-gly-gly-ser.

EXAMPLES Example 1 Construction of 5T4 Sab and Retroviral—VectorDelivery to Tumour

[0200] The cDNA encoding the murine 5T4 monoclonal antibody is clonedand sequenced by standard techniques (Antibody engineering: a practicalapproach ed McCafferty et al. 1996 OUP). The sequence of the variableregion of the antibody can be used to construct a variety ofimmunoglobulin-like molecules including scFvs. The coding sequence of a5T4 scFv, 5T4scFv.1, is shown in FIG. 1a. In this molecule, the DNAsequence encodes the Vh from the mouse 5T4 monoclonal antibody followedby a 15 amino acid flexible linker and the Vl region of the mouse 5T4antibody. The flexible linker encodes 3 copies of the amino-acidsequence gly-gly-gly-gly-ser and the DNA sequence similarity between therepeats has been minimised to avoid the risk of recombination betweenthe repeats when plasmids containing them are grown in E. coli.

[0201] The DNA sequence shown in FIG. 1a can also be used to construct avariety of single-chain antibodies (Sabs) by coupling scFv-encodingsequences to a sequence encoding a Fc region to form an in-frame fusion.A Sab is constructed using a series of DNA cassettes which can beindependently varied to suit particular purposes.

[0202] Cassette 1—Translation Initiation Signal and Signal Peptide

[0203] In order to achieve correct translation initiation and secretionfrom mammalian cells, the following sequence is used:

[0204] aagcttCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCC

[0205] This contains a convenient HindIII restriction site for cloninginto expression vectors (lower case), the consensus translationinitiation signal for mammalian cells (ANNATGPu) and the coding sequencefor a signal peptide sequence from an immunoglobulin gene.

[0206] Cassette 2—scFv

[0207] The sequence of the secreted portion of the 5T4scFv.1 is shown inFIG. 1a. This molecule can be represented as Vh—(gly₄-ser)₃ linker-Vl.

[0208] 5T4 scFv2 consists of the 5T4 variable region sequences connectedin the order Vl—flexible linker Vh. In this case the linker encodes the20 amino-acid peptide (gly₄-ser)₄. A longer linker improves assembly ofthe scFv when the V-region segments are in this order. (Pluckthun et alin Antibody Engineering: a practical approach, ed McCafferty et al. 1996OUP).

[0209] Cassette 3—Heavy Chain Constant Region

[0210] The sequence of a human g1 constant region genomic clone is givenin Ellison et al. 1982 Nucl. Acids Res. 10: 4071-4079. This sequencecontains constant-region introns in addition to the coding sequence.This is fused in-frame to the 3′-end of one of the scFv sequences fromCassette 2. Vectors for convenient assembly of such constructs aredescribed (Walls et al. 1993 Nucl. Acids Res. 21:2921-2929.

[0211] A cDNA of a 5T4 Sab, designated 5T4Sab1 is shown in FIG. 1b,containing cassettes 1, 2 and 3.

[0212] For expression of a 5T4-specific scFv or Sab in human cells, thecoding sequence is inserted into the vector pCIneo (Promega) under thecontrol of a strong promoter and polyadenylation signal. The translationinitiation signal and immunoglobulin leader (signal peptide) sequencefrom Cassette 1 at the 5′ end of the coding region ensure efficientsecretion of the scFv or Sab from mammalian cells.

[0213] For expression of an intact Ig, two separate translationcassettes are constructed, one for the heavy chain and one for the lightchain. These are separated by an internal ribosome—entry site (IRES)from the picornavirus FMDV (Ramesh et al. 1996 Nucl. Acids Res. 24:2697-2700. Alternatively, each cDNA is expressed from a separate copy ofthe hCMV promoter (Ward and Bebbington 1995 In Monoclonal Antibodies edBirch and Lennox.Wiley-Liss).

[0214] For production of retrovirus capable of expressing 5T4 antibodyor immunoglobulin-like molecules with 5T4 specificity, the gene encodinga 5T4-based Sab, or a dicistronic message encoding heavy and lightchains, is inserted into a retroviral vector in which retroviral genomictranscripts are produced from a strong promoter such as the hCMV-MIEpromoter. A suitable plasmid is pHIT111 (Soneoka et al. 1995 Nucl. AcidsRes.23; 628-633) and the required gene is inserted in place of the LacZgene using standard techniques. The resulting plasmid, pHIT-5T4.1 isthen transfected into the FLYRD18 or FLYA13 packaging cell lines (Cossetet al. 1995 J. Virol. 69; 7430-7436) and transfectants selected forresistance to G418 at 1 mg/ml. G418-resistant packaging cells producehigh titres of recombinant retrovirus capable of infecting human cells.The virus preparation is then used to infect human cancer cells and canbe injected into tumours in vivo. The 5T4 Sab is then expressed andsecreted from the tumour cells.

[0215] In pHIT111, the MoMLV LTR promoter-enhancer is used forexpression of the therapeutic gene in the target cell. The vector canalso be modified so that the therapeutic gene is transcribed from aninternal promoter-enhancer such as one which is active predominantly inthe tumour cells or one which contains a hypoxia regulated element. Asuitable promoter is a truncated HSV TK promoter with 3 copies of themouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci. 91: 6496-6500).

Example 2 Transfection of Macrophages/monocytes with an ExpressionVector Encoding TBP

[0216] Peripheral blood mononuclear cells are isolated from humanperipheral blood at laboratory scale by standard techniques procedures(Sandlie and Michaelsen 1996 In Antibody engineering: a practicalapproach. Ed McCafferty et al. Chapter 9) and at large scale byelutriation (eg Ceprate from CellPro). Adherent cells (essentiallymonocytes) are enriched by adherence to plastic overnight and cells canbe allowed to differentiate along the macrophage differentiation pathwayby culturing adherent cells for 1-3 weeks.

[0217] Monocytes and macrophages are transfected with an expressionvector capable of expressing TBP in human cells. For constitutive highlevel expression, the TBP is expressed in a vector which utilises thehCMV-MIE promoter-enhancer, pCI (Promega). For hypoxia-inducedexpression, the hCMV promoter is replaced by a promoter containing atleast one HRE. A suitable promoter is a truncated HSV TK promoter with 3copies of the mouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci.91: 6496-6500).

[0218] A variety of transfection methods can be used to introducevectors into monocytes and macrophages, including particle-mediated DNAdelivery (biolistics), electroporation, cationic agent-mediatedtransfection (eg using Superfect, Qiagen). Each of these methods iscarried out according to the manufacturer's instructions, taking intoaccount the parameters to be varied to achieve optimal results asspecified by the individual manufacturer. Alternatively, viral vectorsmay be used such as defective Adenovirus vectors (Microbix Inc orQuantum Biotechnologies Inc).

Example 3 Assay for ADCC Mediated by Macrophages

[0219] Cells from primary human tumours or tumour cell lines which havebeen transduced with retrovirus expressing TBP are mixed with autologousor heterologous human macrophages, prepared as described in Example 2,for analysis of ADCC activity mediated by the TBP. Alternatively,macrophages engineered to produce TBP as described in Example 2 can beused to direct ADCC on non-transduced tumour cells.

[0220] The assay is carried out according to standard procedures(Sandlie and Michaelsen 1996 In Antibody engineering: a practicalapproach. Ed McCafferty et al. Chapter 9) with appropriatemodifications. Briefly, the effector cells (macrophages or freshlyisolated monocytes) are suspended at 3×10⁶ cells/ml in the appropriatetissue culture medium (DMEM/Hepes, obtained from Life Technologies,containing 1% Foetal Calf Serum). 3×10⁵ tumour target cells, labelledwith ⁵¹Cr are placed in each well of a round-bottomed microtitre platein 0.1 ml culture medium. (Note the culture medium can include spentmedium from the cells producing the TBP). 50 ml effector cells are addedto the wells, the plate is centrifuged at 300 g for 2 min and incubatedat 37° C. for varying periods (eg 4 h) in a tissue culture incubator.The supernatant is then harvested by centrifugation and counted in agamma counter. Results are expressed as percent lysis relative to totalchromium release from an equivalent sample of target cells lysed with0.1% Tween-20. The effector: target cell ratio can be varied in theassay to produce a titration curve.

[0221] For the prior stimulation of macrophage differentiation orpriming, cytokines are added to the cultures. IFNg (Sigma) is added atbetween 100 and 5000 U/ml. CSF-1 or GM-CSF (Santa Cruz Biotechnology)can also be added at appropriate concentrations.

Example 4 Analysis of Efficacy in Animal Models

[0222] Human tumour-derived cell lines and tissues are cultured in vivoin genetically immunodeficient, “nude” mice according to wellestablished techniques (see for example Strobel et al. 1997 Cancer Res.57: 1228-1232; McLeod et al. 1997 Pancreas 14: 237-248). Syngeneic mousemodels, in which a syngeneic tumour line is introduced into animmunocompetent mouse strain may also be used. These serve as suitableanimal models for evaluating gene delivery systems of the invention.Vectors or engineered cells are administered systemically or directlyinto the tumour and tumour growth is monitored in treated and untreatedanimals. This system is used to define the effective dose range of thetreatments of the invention and the most appropriate route ofadministration.

Example 5 Construction of B7—scFv Fusion Proteins

[0223] The extracellular domain of B7-1 is defined by amino-acidresidues 1-215 of the native human B7-1 protein. This sequence, togetherwith its signal peptide-encoding sequence, is used to construct secretedfusion proteins which also contain the scFv derived from the 5T4monoclonal antibody. The sequence of the 5T4 scFv is given in FIG. 1a.

[0224] A DNA coding sequence is constructed using standard molecularbiology techniques which encodes a fusion protein in which theN-terminus of the 5T4 scFv is fused after amino acid 215 of human B7-l.The sequence of this coding sequence, B7-1.5T4.1, is shown in FIG. 2.The fusion protein contains a flexible (gly-gly-gly-gly-ser) spacerbetween the B7-1 and 5T4 scFv sequences. The introduction of aconvenient BmH1 restriction site at the end of the linker insertion(beginning at nucleotide 733) also allows for further linkers to bescreened for optimal expression of bi-functional fusion protein. FIG. 3indicates the fusion protein in diagrammatic form. It is similarlypossible to construct B7-1.5T4.2 (FIG. 3b) in which the scFv isN-terminal and the B7 extracellular domain is C-terminal. In this caseonly the coding sequence of the mature B7-1 (without signal peptide) isrequired. A signal peptide such as an immunoglobulin leader sequence isadded to the N-terminus of the scFv in this instance.

[0225] For fusion proteins which use the co-stimulatory extracellulardomain of B7-2, the signal peptide and extracellular domain of B7-2 isused in place of B7-1 sequences. FIG. 4 shows the coding sequence of theSCM B7-2.5T4.1 co-stimulatory domain. It encodes the first 225 aminoacids of human B7-2, preceded by its signal peptide, and a flexiblelinker (gly4-ser). The BamHI site at the end of this sequence can beused to insert the domain upstream of the 5T4scFv.1 (see FIG. 3). Thesequence includes the B7-2 signal peptide which can serve to allowsecretion of this fusion protein in which the B7-2 domain is at theN-terminus of the fusion protein.

[0226] Each engineered cDNA is inserted into the mammalian expressionvector pCI to allow expression in mammalian tissue culture cells. Forthis purpose, a linker sequence is added to the 5′-end of the codingsequence which introduces a convenient restriction site for insertioninto the polylinker of pCI and adds the translation initiation signalCCACC immediately adjacent to the first ATG codon. Constructs in pCI aretransfected into a suitable mammalian host cell line such as COS-1 toconfirm secretion of the SCM. The transcription cassette from pCI or anappropriate segment of the transcription cassette is subsequentlysub-cloned into the expression vector to be used as the gene deliverysystem for therapeutic use.

Example 6 Transfection of Macrophages/monocytes with an ExpressionVector Encoding an SCM

[0227] Peripheral blood mononuclear cells are isolated from humanperipheral blood at laboratory scale by standard techniques procedures(Sandlie and Michaelsen 1996 In Antibody engineering: a practicalapproach. Ed McCafferty et al. Chapter 9) and at large scale byelutriation (eg Ceprate from CellPro). Adherent cells (essentiallymonocytes) are enriched by adherence to plastic overnight and cells canbe allowed to differentiate along the macrophage differentiation pathwayby culturing adherent cells for 1-3 weeks

[0228] Monocytes and macrophages are transfected with an expressionvector capable of expressing SCM in human cells. For constitutive highlevel expression, the SCM is expressed in a vector which utilises thehCMV-MIE promoter-enhancer, pCI (Promega). For hypoxia-inducedexpression, the hCMV promoter is replaced by a promoter containing atleast one HRE. A suitable promoter is a truncated HSV TK promoter with 3copies of the mouse PGK HRE (Firth et al. 1994 Proc. Natl. Acad. Sci.91: 6496-6500).

[0229] A variety of transfection methods can be used to introducevectors into monocytes and macrophages, including particle-mediated DNAdelivery (biolistics), electroporation, cationic agent-mediatedtransfection (eg using Superfect, Qiagen). Each of these methods iscarried out according to the manufacturer's instructions, taking intoaccount the parameters to be varied to achieve optimal results asspecified by the individual manufacturer. Alternatively, viral vectorsmay be used such as defective Adenovirus vectors (Microbix Inc orQuantum Biotechnologies Inc).

Example 7 Analysis of SCM Binding to CTLA-4 and 5T4-antigen ExpressingCells

[0230] The B7-l or B7-2 domains are expected to bind specifically toCD28 and CTLA-4 present on human T-cells. Binding to T-cells or Chinesehamster ovary cells transfected with human CTLA-4 or CD28 is determinedusing FACS analysis as follows. 5×10⁵ CTLA-4 expressing target cells orequivalent cells lacking CTLA-4 (untransfected CHO cells) are incubatedwith 0.1 ml culture supernatant from COS-1 cells transiently transfectedwith SCM genes for 1 h at 4° C. The cells are washed and incubate with 1mg monoclonal antibody specific for the B7 domain (eg Mab 9E10) followedby FITC-labelled goat anti-mouse IgG (Pharmingen) and analysis by FACS.

[0231] Binding of scFv to 5T4-antigen is similarly assessed using targetcells expressing 5T4-antigen (5T4-transfected A9 cells) or control cells(A9).

Example 8 Analysis of Co-stimulatory Activity

[0232] An established mouse cell line of Balb/c origin such as HC11cells is transfected with the cDNA encoding human 5T4-antigen (Myers etal. 1994 J. Biol. Chem. 269; 9319-9324) inserted in the expressionvector pCIneo.

[0233] Splenic T-cells from Balb/c mice are isolated by standardprocedures (Johnstone and Thorpe 1996 In Immunochemistry in Practice.Blackwell. Chapter 4). T-cells are pre-stimulated by incubation for 1-2days in medium containing 10 ng/ml PMA (Sigma) and 100 U/ml human IL-2(Boehringer Mannheim). HC11-5T4 cells are incubated at 10⁴ cells/well ofa 96-well tissue culture tray for 2 h with up to 0.1 ml supernatant fromCOS cells transfected with SCM gene. Up to 10⁵ pre-stimulated T-cellsare added to each well, the cells are pulsed with 0.25 mCi/well³H-thymidine and incorporation of ³H-thymidine is measured using aliquid scintillation counter after 24 h.

[0234] Incorporation of ³H-thymidine is anticipated to be enhanced bythe presence of SCM.

Example 9 Analysis of Co-stimulation in Animal Models

[0235] HC 11 cells transfected with the human 5T4-antigen gene (Example4) are grown as tumours in Balb/c mice. SCM genes B7-1.5T4.1 orB7-2.5T4.1 or a combination of both genes are introduced into the tumourcells prior to implantation and the growth of the tumours and the growthof control tumours which do not express SCM genes in vivo are monitored.

[0236] It is believed that the expression of SCM genes lead tosignificant reduction in tumour growth.

Example 10 Construction of a B7-1/ScFv, Specific for Human 5T4, FusionProtein

[0237] Standard molecular biology techniques are used to construct afusion protein consisting of the leader sequence and extracellulardomain of B7-1, fused via a flexible linker to the V_(H) and V_(L) ofthe murine Mab 5T4 specific to human 5T4.

[0238] The flexible linker, used to join the extracellular domain ofB7.1 and the ScFv, was constructed by annealing two homologousoligonucleotides with engineered 5′ Sma I and 3′ Spe I sites—usingoligonucleotides

[0239] upper

[0240] 5′ GGG GGT GGT GGG AGC GGT GGT GGC GGC AGT GGC GGC GGC GGA A 3′

[0241] and lower

[0242] 5′ CTA GTT CCG CCG CCG CCA CTG CCG CCA CCA CCG CTC CCA CCA CCC CC3′

[0243] The linker is cloned into pBluescript (Stratagene) via Sma I andSpe I to produce pLINK. The signal peptide (sp) and extracellular domainof murine B7.1 were ampified by PCR from pLK444-mB7.1 (supplied by R.Germain NIH, USA) via primers that introduce 5′ EcoRI and 3′ Sma Isites—primers forward

[0244] 5′ C TCG AAT TCC ACC ATG GCT TGC AAT TGT CAG TTG ATG C 3′

[0245] reverse

[0246] 5′ CTC CCC GGG CTT GCT ATC AGG AGG GTC TTC 3′

[0247] The B7.1 PCR product was cloned into pLINK via Eco RI and Sma Ito form pBS/B7Link.

[0248] The V_(H) and V_(L) of the 5T4 specific ScFv was amplified viaprimers—

[0249] forward primer

[0250] 5′ CTC ACT AGT GAG GTC CAG CTT CAG CAG TC 3′reverse primer

[0251] 5′ CTC GCG GCC GCT TAC CGT TTG ATT TCC AGC TTG GTG CCT CCA CC 3′

[0252] that introduce 5′ Spe I and 3′ Not I sites from pHEN1-5T4 ScFv.PBS/B7Link was digested with Spe I and Not I and ligated with the ScFvto form OBM 233 consisting of the sequence shown as SEQ ID No. 5: B7Link scFv sequence

[0253] This fusion can be used to construct a recombinant vector e.g.retrovirus, Lentivirus, adenovirus, poxvirus, vaccinia virus,baculovirus. Such vectors can be used to inject patient tumoursdirectly. To deliver the fusion protein to tumour cells the recombinantvector is used to transduce macrophages/monocytes/CD34+ cells ex vivobefore injection back into patients. These cells will traffic totumours. The ScFv will bind to a specific tumour antigen expressed onthe surface of tumour cells e.g. 5T4 (Myers et al 1994 JBC). B7 is foundon the surface of professional antigen presenting cells e.g.macrophages, dendritic cells and B cells. It interacts with it ligandsCD28 and CTL-A4 located on CD4 and CD8 cells. The simultaneousinteraction of B7-CD28/CTL-A4 and MHC-peptide/T cell receptor leads to apronounced increase in IL-2 which promotes CD8 (cytotoxic T cell)expansion (Linsley PS, Brady W, Grosmaire L, Aruffo A, Damle N K,Ledbetter J A J Exp Med 1991 Mar 1;173(3):721-730 Binding of the B cellactivation antigen B7 to CD28 costimulates T cell proliferation and I1-2mRNA accumulation.) Tumour cells that have been B7 tranfected with B7have been shown retardation in animal models (Townsend S E, Allison J PScience 1993 15;259(5093):368-370).

Example 11 Transient Expression and Purification of B7-1/ScFv and LScFv

[0254] For transient expression of B7-1/ScFv the human CMV expressionplasmid pCIneo (Promega) was used. B7/ScFv was excised from OBM 233 bydigestion with EcoR I/Not I and cloned into pCIneo that was previouslydigested with EcoRI/Not I. Transient expression of recombinant proteinis made by transfection of 293T cells with the relevant plasmid usingcalcium phosphate (Profectin, Promega). Conditions used were similar tothose recommended by the manufacturer. To reduce bovine serumcontamination serum free optimem media (Gibco BRL). After 36-48 hourstransfection supernatants were harvested and spun through a Centriprep(Amicon, Glos. UK) 10 filter (all proteins larger than 10 kDa arepurified/concentrated) and a Centricon (Amicon) 10 filter. Supernatantsare concentrated approximately 30 fold.

[0255] For B7-1 to be biologically functional it must be able to displaybinding with one of it's natural ligands either CTLA-4 or CD28 found onthe surface of specific populations of T cells (e.g CD4+). Thebiological activity B7-1/ScFv fusion protein was analysed forsimultaneous interaction with its natural ligand CTLA-4 (in the form ofCTLA-4-Ig supplied by Ancell, MN, USA) and A9 cells expressing human5T4. Briefly: approximately 5×10⁵ A9-h5T4 cells were incubated with 100ul of either B7.1 /ScFv or LScFv supernatant in a U bottom 96 well plateat 4° C. for 1 hour. After washing cells were incubated with CTLA-4-Ig(Ancell) for 1 hour. After washing, bound CTLA-4-Ig was detected usingan FITC conjugated anti-mouse Ig (Dako).

[0256] Results show obvious binding of CTLA-Ig with the B7-1extracellular domain, bound via the ScFv, to the surface of human 5T4positive A9 cells. The lack of binding activity with 5T4 negative A9cells further illustrates that the interaction of B7 with CTLA-4-Ig andScFv with 5T4 are specific.

Example 12 ScFv-IgG Fusion Example

[0257] Construction of ScFv-IgG

[0258] The sequence encoding a translation initiation sequence and thehuman immunoglobulin kappa light chain signal peptide is synthesised astwo complementary single stranded oligonucleotides which when annealedalso contain an internal Xho I site at the 5′ end and in addition leavea Xba I compatible 5′ overhang and a Pst I compatible 3′ overhang

[0259] ctagactcgagCCACC ATG GGA TGG AGC TGT ATC ATC CTC TTC TTG GTA GCAACA GCT ACA GGT GTC CAC TCC GAG GTC CAG ctgca

[0260] and

[0261] g CTG GAC CTC GGA GTG GAC ACC TGT AGC TGT TGC TAC CAA GAA GAG GATGAT ACA GCT CCA TCC CAT GGTGGctcgagt

[0262] This is then cloned into pBluescript II (Stratagene) restrictedwith Xba I and Pst I to create pDSII/Leader.

[0263] The 5T4 scFv is amplified by PCR from pHEN1 usingoligonucleotides which incorperate a Pst I site at the 5′ end of theproduct and a Hind III at the 3′ end

[0264] GTC CAG CTG CAG CAG TCT GG

[0265] and

[0266] CG TTT GAT TTC AAG CTT GGT GC

[0267] This is then restricted with those enzymes and inserted intopBSII/Leader restricted with the same enzymes, creatingpBSII/Leader/scFv.

[0268] The HIgG 1 constant region is amplified by PCR from the clonedgene using oligonucleotides which incorperate a Hind III site at the 5′end and a Xho I site at the 3′ end

[0269] gcgc AAG CTT gaa atc aaa cgg GCC TCC ACC AAG GGC CCA

[0270] and

[0271] gcgc ctcgag TCA TTT ACC CGG AGA CAG GG

[0272] This is then restricted with those enzymes and inserted intopBSII/Leader/scFv restricted with the same enzymes, creatingpBSII/Leader/scFv/HG1. The sequence for this construct is shown in theFigures.

[0273] This fusion can be used to construct a recombinant vector e.g.retrovirus, Lentivirus, adenovirus, poxvirus, vaccinia virus,baculovirus. Such vectors can be used to inject patient tumoursdirectly. To deliver the fusion protein to tumour cells the recombinantvector is used to transduce macrophages/monocytes/CD34+ cells ex vivobefore injection back into patients. These cells will traffic totumours. The ScFv will bind to a specific tumour antigen expressed onthe surface of tumour cells e.g. 5T4 (Myers et al 1994 JBC). Bound IgGwill promote specific tumour destruction via a collection of mechanismscollectively known as antibody dependent cellular cytotoxicity (Munn etal Can res 1991 ibid, Primus et al 1993 Cancer Res ibid).

Example 13 Construction of ScFv-IgE1 (Human IgE1 Heavy Constant Region)

[0274] A similar fusion construct of 5T4 scFv—human IgE constant heavychain is made consisting of the sequence shown as SEQ ID No. 6.

[0275] This fusion construct is made by amplifying the human IgE1constant heavy region by PCR cDNA derived from human B-cells RNA by RTand subsequently using oligonucleotides which incorporate a Hind IIIsite at the 5′ end and a Xho I site at the 3′ end

[0276] gcgc AAG CTT gaa atc aaa cgg GCC TCC ACA CAG AGC CCA and

[0277] gcgc ctcgag TCA TTT ACC GGG ATT TAC AGA

[0278] This is then restricted with those enzymes and inserted intopBSII/Leader/scFv restricted with the same enzymes, creatingpBSII/Leader/scFv/DIE1.

[0279] As described above the ScFv-IgE construct can be incorporatedinto a recombinant viral vector for use in gene therapy of cancer e.g.inject patient tissue directly or to transduce patient derivedmacrophages/monocytes/CD34+ cells ex vivo. The fusion protein will besecreted and will bind to tumour cells bearing the antigen that the ScFvis specific for. Binding of IgE to tumour cells should promote a stronghistamine response via activation of mast cells. This will lead to astrong inflammatory response and destruction tumour cells as is reportedfor IgE cytotoxic destruction of parasites e.g. helminth larvae (CapronM 1988 Eosinophils in diseases: receptors and mediators. In progress inallergy and clinical immunology (Proc. 13^(th) Int. Congress of Allergyand Clinical Immunology) Hogrefe & Huber Toronto p6). Such inflammationand tumour destruction should initiate the recruitment of other immuneeffector cells. Past reports indicate that treatment with an MMTVantigen specific IgE Mab leads to protection from a tumour expressingMMTV antigen (Nagy E Istanvan B, Sehon A H 1991 Cancer Immunol.Immunotherapy vol 34:63-69).

Example 14 Construction of B7/EGF

[0280] B7—EGF Synthetic Gene

[0281] A fusion construct of B7—EGF is made by inserting a PCR productamplified from the region of the gene encoding the mature EGF peptide(see accession number X04571) into pBS/B7 Link. This construct has thesequence shown as SEQ ID No. 7.

[0282] Using cDNA derived by RT of RNA isolated from a cell line suchthe 293 human kidney line (ATCC: CRL1573), the DNA is amplified by PCRusing oligonucleotides containing a Spe I restriction enzyme site at theN-terminus and a stop codon and a Not I site at the C-terminus

[0283] GG ACT AGT AAT AGT GAC TCT GAA TGT CCC

[0284] and

[0285] ATT AGC GGC CGC TTA GCG CAG TTC CCA CCA CTT C

[0286] The resulting product is digested with those enzymes and ligatedto pBS/B7 Link which has been restricted -with the same enzymes creatingpBS/B7 Link EGF. The B7 Link EGF cassette is then excised with Eco RIand Not I and inserted into a derivative of pHIT111 (Soneoka et al,1995, Nucl Acid Res 23; 628) which no longer carries the LacZ gene

[0287] An alternative to using ScfV is to use growth factors that have ahigh affinity to their corresponding receptor e.g. epidermal growthfactor which binds to several receptors including erb-2 which is highlyassociated with tumourgenesis.

[0288] As described above the fusion construct can be incorporated intoa recombinant viral vector for use in gene therapy e.g. inject patienttissue directly or to transduce patient derivedmacrophages/moncytes/CD34+ cells ex vivo. The fusion protein will besecreted and will bind to tumour cells bearing the erb-2 antigen.

[0289] Epidermal growth factor (EGF) will bind to its ligand erb-2 (anEGF receptor) thus obviating the requirement of a ScFv. Erb-2 is highlyassociated with tumour cells (Hynes N E Semin Cancer Biol 1993Feb;4(1):19-26, Amplification and over expression of the erbB-2 gene inhuman tumors: its involvement in tumor development, significance as aprognostic factor, and potential as a target for cancer therapy). B7 isfound on the surface of professional antigen presenting cells e.g.macrophages, dendritic cells and B cells. It interacts with it ligandsCD28 and CTL-A4 located on CD4 and CD8 cells. The simultaneousinteraction of B7-CD28/CTL-A4 and MHC-peptide/T cell receptor leads tomassive increase in IL-2 which promotes CD8 (cytotoxic T cell) expansion(Linsley P S, Brady W, Grosmaire L, Aruffo A, Damle N K, Ledbetter J A JExp Med 1991 March 1;173(3):721-730 Binding of the B cell activationantigen B7 to CD28 costimulates T cell proliferation and interleukin 2mRNA accumulation.) Tumour cells that have been B7 transfected with B7have shown retardation in animal models (Townsend S E, Allison J PScience 1993 15;259(5093):368-370 Tumor rejection after directcostimulation of CD8+ T cells by B7-transfected melanoma cells). It ishas been reported that B7 will enhance the CTL response to tumourantigens specific to tumour cells thus leading to the destruction of allsuch cells.

Example 15 Production of Cell Lines Expressing Fusion Constructs

[0290] The ScFv-IgG gene was excised from pBSII/L/ScFv/hIgG1 by Xho Idigestion, and cloned into pLXSN via the Xho I site, to makepLXSN/ScFv-IgG, such that after chromosomal integration it is undertranscriptional control of the LTR. Virus was made in the human kidneycell line 293T by co-transfecting plasmids containing the MLV gap-polgenes (pCIEGPPD) and and the VSV G envelope (pRV67) using the tripleplasmid HIT system (Landau & Littman 1992 J Virol 66 5110, Soneoka Y etal 1995 NAR 23:628-633). Virus is harvested after 48 hours and used totransduce BHK-21 cells (ATCC# CCL-10). Approximately 24 hourspost-transduction, transduced cells are selected by the addition of 1mg/ml G418 (Gibco BRL) to culture medium. The supernatant from positivecolonies was harvested and concentrated by centrifugation through aCentriprep (Amicon, Glos. UK) 10 filter (all proteins larger than 10 kDaare purified/concentrated) and a Centricon (Amicon) 10 filter.Supernatants were concentrated approximately 30 fold.

[0291] Other fusion proteins are cloned into pLXSN via the Xho I siteand expressed and concentrated using a similar protocol.

[0292] FACS analysis of fusion protein binding with cells expressingspecific ligand To determine if the ScFv-IgG fusion protein is specificfor its antigen, human 5T4, FACS analysis of a human bladder carcinomatumour line (EJ) or a stable murine cell line expressing h5T4, A9-h5T4(Myers et al 1994 JBC) and a 5T4 negative line A9-neo was carried out.Approximately 5×10⁵ A9 or EJ cells, in a round bottom 96 well plate(Falcon) were incubated with 100 ul of a 1:5 dilution of concentratedsupernatant (as described above) for 1 hour at 4° C. After washing,bound protein is detected using an anti human IgG/FITC conjugatedantibody (Dako). Cells were analysed on a Becton Dickinson FACS machine.FACS results show that there is at least a 1 log shift in fluorescenceactivity in those 5T4 positive cells treated with the ScFv-IgG constructcompared to the negative control construct consisting of the ScFvprotein alone. A9 neo FACS shows that there is no non-specific bindingof the ScFv component of the fusion protein.

[0293] FACS analysis of ScFv-IgE is carried out similar to above exceptthat anti-human IgE-FITC (Dako) is used to detect binding of the fusionprotein.

[0294] The B7/EGF fusion protein is analysed for binding using FACS andHC11-erb-2 positive cells (Hynes et al 1990). CTLA-4-Ig (Ancell, USA) isused to analyse the bioactivity of the B7 component of the bound fusionprotein. Anti-mouse IgG-FITC is used to show CTLA-4 binding.

SUMMARY

[0295] The present invention therefore provides a means for delivering,for example, therapeutic compounds to a tumour site.

[0296] All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

1 24 1 910 DNA Artificial Sequence Coding sequence of a 5T4scFvdesignated 5T4scFv.1. 1 gaggtccagc ttcagcagtc tggacctgac ctggtgaagcctggggcttc vsgdvkgasa 60 gtgaagatat cctgcaaggc ttctggttac tcattcactggctactacav ksckasgyst 120 gyytgcactg ggtgaagcag agccatggaa agagccttgagtggattgga cgtmhwvksh 180 gkswgratta atcctaacaa tggtgttact ctctacaaccagaaattcaa ggacaannng 240 vtynkkdkgg ccatattaac tgtagacaag tcatccaccacagcctacat ggagctccat 300 vdkssttaym gcagcctgac atctgaggac tctgcggtctattactgtgc aagatctact 360 rstsdsavyy carstatgat tacgaactat gttatggactactggggtca agtaacctca 420 gtcacmtnyv mdywgvtsvt cgtctcctca ggtggtggtgggagcggtgg tggcggcact 480 ggcggcggcg vssggggsgg ggtggggatc tagtattgtgatgacccaga ctcccacatt 540 cctgcttgtt tcagcagssv mtttvsagga gacagggttaccataacctg caaggccagt 600 cagagtgtga gtaatgagdr vttckassvs ndtgtagdttggtaccaaca gaagccaggg 660 cagtctccta cactgctcat atvawykgst cctatacatccagtcgctac gctggagtcc 720 ctgatcgctt cattggcagt sytssryagv drgsggatatgggacggatt tcactttcac 780 catcagcact ttgcaggctg aagagygtdt tstadcctggcagtttattt ctgtcagcaa 840 gattataatt ctcctccgac gttcgavycd ynstgtggaggcaccaagct ggaaatcaaa 900 cgggggtkkr 910 2 2239 DNA Artificial SequenceCoding sequence of a 5T4scFv designated 5T4scFv1. 2 aagcttccaccatgggatgg agctgtatca tcctcttctt ggtagcaaca astmgwscva 60 tgctacaggtgtccactccg aggtccagct tcagcagtct ggacctgacc tatgvhsvsg 120 dggtgaagcctggggcttca gtgaagatat cctgcaaggc ttctggttac tvkgasvksc 180 kasgycattcactggctact acatgcactg ggtgaagcag agccatggaa agagcstgyy 240 mhwvkshgkscttgagtgga ttggacgtat taatcctaac aatggtgtta ctctctacaa 300 wgrnnngvtynccagaaatt caaggacaag gccatattaa ctgtagacaa gtcatccacc 360 akkdkatvdksstcagccta catggagctc cgcagcctga catctgagga ctctgcggtc 420 tattaymrstsdsavytact gtgcaagatc tactatgatt acgaactatg ttatggacta 480 ctggggycarstmtnyvmdy wgtcaagtaa cctcagtcac cgtctcctca ggtggtggtg 540 ggagcggtggtgvtsvtvss ggggsgggcg gcactggcgg cggcggatct agtattgtga 600 tgacccagactcccacaggt ggggssvmtt tttcctgctt gtttcagcag gagacagggt 660 taccataacctgcaaggcca gvsagdrvtt ckastcagag tgtgagtaat gatgtagctt 720 ggtaccaacagaagccaggg cagtsvsndv awykgctcct acactgctca tatcctatac 780 atccagtcgctacgctggag tccctstsyt ssryagvgat cgcttcattg gcagtggata 840 tgggacggatttcactttca ccatcagdrg sgygtdttsc actttgcagg ctgaagacct 900 ggcagtttatttctgtcagc aagattatat adavycdyat tctcctccga cgttcggtgg 960 aggcaccaagctggaaatca aacgggccns tgggtkkrat ccaccaaggg cccatcggtc 1020 ttccccctggcaccctcctc caagagcacs tkgsvassks tctctggggg cacagcggcc 1080 ctgggctgcctggtcaagga ctacttcccc gsggtaagcv kdyaaccggt gacggtgtcg 1140 tggaactcaggcgccctgac cagcggcgtg cacvtvswns gatsgvhacc ttcccggctg 1200 tcctacagtcctcaggactc tactccctca gcagcgttav ssgysssvgg tgaccgtgcc 1260 ctccagcagcttgggcaccc agacctacat ctgcaacgvt vsssgttycn tgaatcacaa 1320 gcccagcaacaccaaggtgg acaagaaagt tgagcccaaa vnhksntkvd kkvktcttgt 1380 gacaaaactcacacatgccc accgtgccca gcacctgaac tcctscdkth tccagggggg 1440 accgtcagtcttcctcttcc ccccaaaacc caaggacacc ctcaggsvkk dttgatctcc 1500 cggacccctgaggtcacatg cgtggtggtg gacgtgagcc acmsrtvtcv vvdvshgaag 1560 accctgaggtcaagttcaac tggtacgtgg acggcgtgga ggtgcadvkn wyvdgvvhta 1620 atgccaagacaaagccgcgg gaggagcagt acaacagcac gtaccgtgna ktkrynstyr 1680 tggtcagcgtcctcaccgtc ctgcaccagg actggctgaa tggcaaggag vvsvtvhdwn 1740 gktacaagtgcaaggtctcc aacaaagccc tcccagcccc catcgagaaa acykckvsnk 1800 aaktcatctccaaagccaaa gggcagcccc gagaaccaca ggtgtacacc ctgcskakgr 1860 vytccccatcccgggatgag ctgaccaaga accaggtcag cctgacctgc ctgsrdtknv 1920 stcgtcaaaggcttctatcc cagcgacatc gccgtggagt gggagagcaa tggvkgysda 1980 vwsnggcagccggagaacaa ctacaagacc acgcctcccg tgctggactc cgacgnnykt 2040 tvdsdgctccttcttcctct acagcaagct caccgtggac aagagcaggt ggcaggsysk 2100 tvdksrwcaggggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccagnv 2160 scsvmhahnhctacacgcag aagagcctct ccctgtctcc gggtaaatga gtgcgacggc 2220 ytksssgkvrrcaagctts 2239 3 1809 DNA Artificial Sequence Coding sequence ofB7-1.5T4.1 3 atgggccaca cacggaggca gggaacatca ccatccaagt gtccatacctmghtrrgtss 60 kcycaatttc tttcagctct tggtgctggc tggtctttct cacttctgttcagnvagshc 120 sgtgttatcc acgtgaccaa ggaagtgaaa gaagtggcaa cgctgtcctgtgvhvtkvkv 180 atscggtcac aatgtttctg ttgaagagct ggcacaaact cgcatctactggcaghnvsv 240 atrywaaagg agaagaaaat ggtgctgact atgatgtctg gggacatgaatatatkkkmv 300 tmmsgdmngg cccgagtaca agaaccggac catctttgat atcactaataacctctccwy 360 knrtdtnnsa ttgtgatcct ggctctgcgc ccatctgacg agggcacatacgagtgtgtv 420 arsdgtycvt gttctgaagt atgaaaaaga cgctttcaag cgggaacacctggctgaagv 480 kykdakrhat gacgttatca gtcaaagctg acttccctac acctagtatatctgactttv 540 tsvkadtssd gaaattccaa cttctaatat tagaaggata atttgctcaacctctggagg 600 tsnrrcstsg gttttccaga gcctcacctc tcctggttgg aaaatggagaagaattaaat 660 ghswngncca tcaacacaac agtttcccaa gatcctgaaa ctgagctctatgctgttant 720 tvsdtyavag cagcaaactg gatttcaata tgacaaccaa ccacagcttcatgtgtctss 780 kdnmttnhsm ccatcaagta tggacattta agagtgaatc agaccttcaactggaataca 840 akyghrvntn wntccaagca agagcatttt cctgatggag gcgggggatccgaggtccag 900 ctttkhdggg gsvcagcagt ctggacctga cctggtgaag cctggggcttcagtgaagat 960 atcsgdvkga svksctgcaa ggcttctggt tactcattca ctggctactacatgcactgg 1020 gtgackasgy stgyymhwva gcagagccat ggaaagagcc ttgagtggattggacgtatt 1080 aatcctaack shgkswgrnn aatggtgtta ctctctacaa ccagaaattcaaggacaagg 1140 ccatattaac ngvtynkkdk attgtagaca agtcatccac cacagcctacatggagctcc 1200 gcagcctgac atvdksstta ymrstctgag gactctgcgg tctattactgtgcaagatct 1260 actatgatta cgaacsdsav yycarstmtn tatgttatgg actactggggtcaagtaacc 1320 tcagtcaccg tctcctcagg yvmdywgvts vtvssgtggt ggtgggagcggtggtggcgg 1380 cactggcggc ggcggatcta gtattggggs ggggtggggs stgatgacccagactcccac 1440 attcctgctt gtttcagcag gagacagggt tvmtttvsag drvaccataacctgcaaggc 1500 cagtcagagt gtgagtaatg atgtagcttg gtattckass vsndvawyccaacagaagcc 1560 agggcagtct cctacactgc tcatatccta tacatccakg stsytsgtcgctacgctgga 1620 gtccctgatc gcttcattgg cagtggatat gggacgsrya gvdrgsgygtgatttcactt 1680 tcaccatcag cactttgcag gctgaagacc tggcagttta dttstadavytttctgtcag 1740 caagattata attctcctcc gacgttcggt ggaggcacca cdynstgggtagctggaaat 1800 caaataakk 1809 4 887 DNA Artificial Sequence Human B7-2sequence followed by a linker. 4 atgggactga gtaacattct ctttgtgatggccttcctgc tctctggtgc mgsnvmasga 60 tgctcctctg aagattcaag cttatttcaatgagactgca gacctgccat akayntadgc 120 caatttgcaa actctcaaaa ccaaagcctgagtgagctag tagtatttca nsnssvvtgg 180 caggaccagg aaaacttggt tctgaatgaggtatacttag gcaaagawdn vnvygkgaaa 240 tttgacagtg ttcattccaa gtatatgggccgcacaagtt ttgattkdsv hskymgrtsd 300 cggacagttg gaccctgaga cttcacaatcttcagatcaa ggacaagggc sdswtrhnkd 360 kgttgtatca atgtatcatc catcacaaaaagcccacagg aatgattcgc atychhkktg 420 mrccaccaga tgaattctga actgtcagtgcttgctaact tcagtcaacc tghmnssvan 480 saaatagtac caatttctaa tataacagaaaatgtgtaca taaatttgac cvsntnvynt 540 tgctcatcta tacacggtta cccagaacctaagaagatga gtgttttgct csshgykkms 600 vaagaaccaa gaattcaact atcgagtatgatggtattat gcagaaatct crtknstydg 660 mksaagataa tgtcacagaa ctgtacgacgtttccatcag cttgtctgtt tcadnvtydv 720 sssvsttccc tgatgttacg agcaatatgaccatcttctg tattctggaa actgadvtsn 780 mtctdcaaga cgcggctttt atcttcacctttctctatag agcttgagga ccctcktrss 840 sdagcctccc ccagaccaca ttcctggaggcgggggatcc dhggggs 887 5 1518 DNA Artificial SequencepBSII/Leader/scFv/HG1. 5 atggcttgca attgtcagtt gatgcaggat acaccactcctcaagtttcc atgtccaagg 60 ctcattcttc tctttgtgct gctgattcgt ctttcacaagtgtcttcaga tgttgatgaa 120 caactgtcca agtcagtgaa agataaggta ttgctgccttgccgttacaa ctctccgcat 180 gaagatgagt ctgaagaccg aatctactgg caaaaacatgacaaagtggt gctgtctgtc 240 attgctggga aactaaaagt gtggcccgag tataagaaccggactttata tgacaacact 300 acctactctc ttatcatcct gggcctggtc ctttcagaccggggcacata cagctgtgtc 360 gttcaaaaga aggaaagagg aacgtatgaa gttaaacacttggctttagt aaagttgtcc 420 atcaaagctg acttctctac ccccaacata actgagtctggaaacccatc tgcagacact 480 aaaaggatta cctgctttgc ttccgggggt ttcccaaagcctcgcttctc ttggttggaa 540 aatggaagag aattacctgg catcaatacg acaatttcccaggatcctga atctgaattg 600 tacaccatta gtagccaact agatttcaat acgactcgcaaccacaccat taagtgtctc 660 attaaatatg gagatgctca cgtgtcagag gacttcacctgggaaaaacc cccagaagac 720 cctcctgata gcaagcccgg gggtggtggg agcggtggtggcggcagtgg cggcggcgga 780 actagtgagg tccagcttca gcagtctgga cctgacctggtgaagcctgg ggcttcagtg 840 aagatatcct gcaaggcttc tggttactca ttcactggctactacatgca ctgggtgaag 900 cagagccatg gaaagagcct tgagtggatt ggacgtattaatcctaacaa tggtgttact 960 ctctacaacc agaaattcaa ggacaaggcc atattaactgtagacaagtc atccaccaca 1020 gcctacatgg agctccgcag cctgacatct gaggactctgcggtctatta ctgtgcaaga 1080 tctactatga ttacgaacta tgttatggac tactggggtcaagtaacttc agtcaccgtc 1140 tcttcaggtg gtggtgggag cggtggtggc ggcactggcggcggcggatc tagtattgtg 1200 atgacccaga ctcccacatt cctgcttgtt tcagcaggagacagggttac cataacctgc 1260 aaggccagtc agagtgtgag taatgatgta gcttggtaccaacagaagcc agggcagtct 1320 cctacactgc tcatatccta tacatccagt cgctacgctggagtccctga tcgcttcatt 1380 ggcagtggat atgggacgga tttcactttc accatcagcactttgcaggc tgaagacctg 1440 gcagtttatt tctgtcagca agattataat tctcctccgacgttcggtgg aggcaccaag 1500 ctggaaatca aacggtaa 1518 6 2090 DNAArtificial Sequence 5T4 scFv-human IgE fusion construct. 6 ctcgagccaccatgggatgg agctgtatca tcctcttctt ggtagcaaca gctacaggtg 60 tccactccgaggtccagctg cagcagtctg gacctgacct ggtgaagcct ggggcttcag 120 tgaagatatcctgcaaggct tctggttact cattcactgg ctactacatg cactgggtga 180 agcagagccatggaaagagc cttgagtgga ttggacgtat taatcctaac aatggtgtta 240 ctctctacaaccagaaattc aaggacaagg ccatattaac tgtagacaag tcatccacca 300 cagcctacatggagctccgc agcctgacat ctgaggactc tgcggtctat tactgtgcaa 360 gatctactatgattacgaac tatgttatgg actactgggg tcaagtaact tcagtcaccg 420 tctcttcaggtggtggtggg agcggtggtg gcggcactgg cggcggcgga tctagtattg 480 tgatgacccagactcccaca ttcctgcttg tttcagcagg agacagggtt accataacct 540 gcaaggccagtcagagtgtg agtaatgatg tagcttggta ccaacagaag ccagggcagt 600 ctcctacactgctcatatcc tatacatcca gtcgctacgc tggagtccct gatcgcttca 660 ttggcagtggatatgggacg gatttcactt tcaccatcag cactttgcag gctgaagacc 720 tggcagtttatttctgtcag caagattata attctcctcc gacgttcggt ggaggcacca 780 agcttgaaatcaaacgggcc tccacacaga gcccatccgt cttccccttg acccgctgct 840 gcaaaaacattccctccaat gccacctccg tgactctggg ctgcctggcc acgggctact 900 tcccggagccggtgatggtg acctgggaca caggctccct caacgggaca actatgacct 960 taccagccaccaccctcacg ctctctggtc actatgccac catcagcttg ctgaccgtct 1020 cgggtgcgtgggccaagcag atgttcacct gccgtgtggc acacactcca tcgtccacag 1080 actgggtcgacaacaaaacc ttcagcgtct gctccaggga cttcaccccg cccaccgtga 1140 agatcttacagtcgtcctgc gacggcggcg ggcacttccc cccgaccatc cagctcctgt 1200 gcctcgtctctgggtacacc ccagggacta tcaacatcac ctggctggag gacgggcagg 1260 tcatggacgtggacttgtcc accgcctcta ccacgcagga gggtgagctg gcctccacac 1320 aaagcgagctcaccctcagc cagaagcact ggctgtcaga ccgcacctac acctgccagg 1380 tcacctatcaaggtcacacc tttgaggaca gcaccaagaa gtgtgcagat tccaacccga 1440 gaggggtgagcgcctaccta agccggccca gcccgttcga cctgttcatc cgcaagtcgc 1500 ccacgatcacctgtctggtg gtggacctgg cacccagcaa ggggaccgtg aacctgacct 1560 ggtcccgggccagtgggaag cctgtgaacc actccaccag aaaggaggag aagcagcgca 1620 atggcacgttaaccgtcacg tccaccctgc cggtgggcac ccgagactgg atcgaggggg 1680 agacctaccagtgcagggtg acccaccccc acctgcccag ggccctcatg cggtccacga 1740 ccaagaccagcggcccgcgt gctgccccgg aagtctatgc gtttgcgacg ccggagtggc 1800 cggggagccgggacaagcgc accctcgcct gcctgatcca gaacttcatg cctgaggaca 1860 tctcggtgcagtggctgcac aacgaggtgc agctcccgga cgcccggcac agcacgacgc 1920 agccccgcaagaccaagggc tccggcttct tcgtcttcag ccgcctggag gtgaccaggg 1980 ccgaatgggagcagaaagat gagttcatct gccgtgcagt ccatgaggca gcgagcccct 2040 cacagaccgtccagcgagcg gtgtctgtaa atcccggtaa atgagagctc 2090 7 945 DNA ArtificialSequence B7-EGF fusion construct. 7 atggcttgca attgtcagtt gatgcaggatacaccactcc tcaagtttcc atgtccaagg 60 ctcattcttc tctttgtgct gctgattcgtctttcacaag tgtcttcaga tgttgatgaa 120 caactgtcca agtcagtgaa agataaggtattgctgcctt gccgttacaa ctctccgcat 180 gaagatgagt ctgaagaccg aatctactggcaaaaacatg acaaagtggt gctgtctgtc 240 attgctggga aactaaaagt gtggcccgagtataagaacc ggactttata tgacaacact 300 acctactctc ttatcatcct gggcctggtcctttcagacc ggggcacata cagctgtgtc 360 gttcaaaaga aggaaagagg aacgtatgaagttaaacact tggctttagt aaagttgtcc 420 atcaaagctg acttctctac ccccaacataactgagtctg gaaacccatc tgcagacact 480 aaaaggatta cctgctttgc ttccgggggtttcccaaagc ctcgcttctc ttggttggaa 540 aatggaagag aattacctgg catcaatacgacaatttccc aggatcctga atctgaattg 600 tacaccatta gtagccaact agatttcaatacgactcgca accacaccat taagtgtctc 660 attaaatatg gagatgctca cgtgtcagaggacttcacct gggaaaaacc cccagaagac 720 cctcctgata gcaagcccgg gggtggtgggagcggtggtg gcggcagtgg cggcggcgga 780 actagtaata gtgactctga atgtcccctgtcccacgatg ggtactgcct ccatgatggt 840 gtgtgcatgt atattgaagc attggacaagtatgcatgca actgtgttgt tggctacatc 900 ggggagcgat gtcagtaccg agacctgaagtggtgggaac tgcgc 945 8 47 DNA Artificial Sequence Oligonucleotide. 8ctagttccgc cgccgccact gccgccacca ccgctcccac caccccc 47 9 38 DNAArtificial Sequence Forward primer. 9 ctcgaattcc accatggctt gcaattgtcagttgatgc 38 10 30 DNA Artificial Sequence Reverse primer. 10 ctccccgggcttgctatcag gagggtcttc 30 11 29 DNA Artificial Sequence Forward primer.11 ctcactagtg aggtccagct tcagcagtc 29 12 44 DNA Artificial SequenceReverse primer. 12 ctcgcggccg cttaccgttt gatttccagc ttggtgcctc cacc 4413 87 DNA Artificial Sequence Oligonucleotide containing translationinitiation site and signal peptide. 13 ctagactcga gccaccatgg gatggagctgtatcatcctc ttcttggtag caacagctac 60 aggtgtccac tccgaggtcc agctgca 87 1479 DNA Artificial Sequence Oligonucleotide containing translationinitiation site and signal peptide. 14 gctggacctc ggagtggaca cctgtagctgttgctaccaa gaagaggatg atacagctcc 60 atcccatggt ggctcgagt 79 15 20 DNAArtificial Sequence Primer with PstI site. 15 gtccagctgc agcagtctgg 2016 22 DNA Artificial Sequence Primer with Hind III site. 16 cgtttgatttcaagcttggt gc 22 17 40 DNA Artificial Sequence Primer for the constantregion which incorporates a Hind III site. 17 gcgcaagctt gaaatcaaacgggcctccac caagggccca 40 18 30 DNA Artificial Sequence Primer for theconstant region which incorporates a XhoI site. 18 gcgcctcgag tcatttacccggagacaggg 30 19 40 DNA Artificial Sequence Oligonucleotide with HindIIIsite. 19 gcgcaagctt gaaatcaaac gggcctccac acagagccca 40 20 31 DNAArtificial Sequence Oligonucleotide with XhoI site. 20 gcgcctcgagtcatttaccg ggatttacag a 31 21 29 DNA Artificial Sequence Oligonuclotidewith SpeI site. 21 ggactagtaa tagtgactct gaatgtccc 29 22 34 DNAArtificial Sequence Oligonucleotide with NotI site and Stop codon. 22attagcggcc gcttagcgca gttcccacca cttc 34 23 68 DNA Artificial SequenceTranslation initiation and secretion signal. 23 aagcttccac catgggatggagctgtatca tcctcttctt ggtagcaaca gctacaggtg 60 tccactcc 68 24 43 DNAArtificial Sequence Coding sequence of a 5T4scFv designated 5T4scFv.1.24 gggggtggtg ggagcggtgg tggcggcagt ggcggcggcg gaa 43

What is claimed is:
 1. A vector comprising a nucleotide sequence codingfor a tumor interacting protein, and optionally comprising a nucleotidesequence of interest which encodes a product of interest; wherein thetumor interacting protein is capable of recognizing a tumor, such thatthe vector is capable of delivering the nucleotide sequence of interestand/or the product of interest to the tumor, and wherein the tumorinteracting protein comprises an antibody comprising at least one 5T4antibody complementarity-determining region (CDR).
 2. The vectoraccording to claim 1 wherein the CDR is a CDR of the 5T4 ScFv antibodyamino acid sequence comprising SEQ ID NO:1 or of the 5T4 Sab antibodyamino acid sequence comprising SEQ ID NO:2.
 3. The vector according toclaim 2 wherein the CDR comprises an amino acid sequence selected fromthe group consisting of KASQSVSNDVA, YTSSRYA, QQDYNSPPT, GYYMH,RINPNNGVTLYNQKFKD, and STMITNYVMDY.
 4. The vector according to claim 1wherein the antibody is a 5T4 Fab, 5T4 Fv or 5T4 ScFv fragment.
 5. Thevector according to claim 4 wherein the antibody is the 5T4 ScFvfragment having the amino acid sequence of SEQ ID NO:
 1. 6. The vectoraccording to claim 1 wherein the antibody comprises an ScFv fragmentcoupled to an Fc region.
 7. The vector according to claim 6 wherein theantibody comprises the 5T4 Sab antibody amino acid sequence of SEQ IDNO:
 2. 8. The vector according to claim 1 wherein the antibody is ahumanized antibody.
 9. The vector according to claim 8 wherein theantibody is an intact humanized antibody.
 10. The vector according toclaim 8 wherein the antibody is a humanized single chain antibody. 11.The vector according to claim 10 wherein the antibody comprises an ScFvfragment coupled to an Fc region.
 12. The vector according to claim 8wherein the CDR comprises an amino acid sequence selected from the groupconsisting of KASQSVSNDVA, YTSSRYA, QQDYNSPPT, GYYMH, RINPNNGVTLYNQKFKDand STMITNYVMDY.
 13. A vector comprising a nucleotide sequence codingfor a tumor interacting protein, and optionally comprising a nucleotidesequence of interest which encodes a product of interest; wherein thetumor interacting protein is capable of recognizing a tumor, such thatthe vector is capable of delivering the nucleotide sequence of interestand/or the product of interest to the tumor, and wherein the vector iscapable of expressing the tumor interacting protein in a mammalian cell,and wherein the tumor interacting protein comprises an antibodycomprising at least one 5T4 antibody complementarity-determining region(CDR).
 14. A CDR-grafted antibody comprising an antibody comprising atleast one 5T4 antibody complementarity-determining region (CDR).
 15. Theantibody according to claim 14 wherein the CDR is a CDR of a 5T4 ScFvantibody amino acid sequence of SEQ ID NO:1 or is a CDR of a 5T4 Sabantibody amino acid sequence of SEQ ID NO:2.
 16. The antibody accordingto claim 15 wherein the CDR comprises an amino acid sequence selectedfrom the group consisting of KASQSVSNDVA, YTSSRYA, QQDYNSPPT, GYYMH,RINPNNGVTLYNQKFKD, and STMITNYVMDY.
 17. The antibody according to claim16 wherein the antibody is a 5T4 Fab, a 5T4 Fv or a 5T4 ScFv fragment.18. The antibody according to claim 17 wherein the antibody is a 5T4ScFv fragment having the amino acid sequence of SEQ ID NO:1.
 19. Theantibody according to claim 18 wherein the antibody comprises an ScFvfragment coupled to an Fc region.
 20. The antibody according to claim 18wherein the antibody comprises the 5T4 Sab antibody amino acid sequenceof SEQ ID NO:2.
 21. The antibody according to claim 14 wherein theantibody is a humanized antibody.
 22. The antibody according to claim 21wherein the antibody is an intact humanized antibody.
 23. The antibodyaccording to claim 21 wherein the antibody is a humanized single chainantibody.
 24. The antibody according to claim 23 wherein the antibodycomprises an ScFv fragment coupled to an Fc region.
 25. The antibodyaccording to claim 21 wherein the CDR comprises an amino acid sequenceselected from the group consisting of KASQSVSNDVA, YTSSRYA, QQDYNSPPT,GYYMH, RINPNNGVTLYNQKFKD and STMITNYVMDY.